【摘要】 目的:研究聚酰胺(polyamidoamine,PAMAM)树形分子高聚合物作为survivin反义寡核苷酸(survivin antisense oligodeoxynucleotide,survivin-asODN)载体传递系统的可行性以及对肝癌SMMC-7721细胞凋亡的影响。方法:将200μg/Lsurvivin-asODN和3.66μg/L PAMAM混合制备PAMAM反义基因复合物,同时制备阳离子脂质体反义基因复合物作为对照。透射电镜观察复合物的形态、粒径,Zeta电位分析仪测定复合物的zeta电位,离心法和紫外分光光度仪测定复合物的包封率、载药率和体外DNA释放速度。将上述两种基因载体复合物转染肝癌SMMC-7721细胞,测定其转染效率;Western blotting检测转染后肿瘤细胞中survivin蛋白的表达;流式细胞术检测肿瘤细胞的凋亡率。结果:成功制备PAMAM反义基因载体系统PAMAM-survivin-asODN。该复合物的粒径小于脂质体-survivin-asODN复合物的粒径[(64.9±9.60)vs(193.9±32.20)nm,P<0.01],但zeta电位高于后者[(37.7±3.80)vs(22.6±2.20)mV,P<0.05];两者基因载药率、包封率无显著差异;PAMAM反义基因复合物对DNA持续释放达14 d,但脂质体复合物只持续5 d。PAMAM-survivin-asODN转染肝癌细胞的效果强于脂质体-survivin-asODN(P<0.05)。转染后肝癌细胞siurvivin蛋白的表达显著低于脂质体复合物[(26.80±5.65)vs(36.96±5.89),P<0.05],该细胞的凋亡率显著高于脂质体复合物转染细胞[(60.3±8.25)%vs(48.7±9.39)%,P<0.05]。结论:PAMAM作为载体系统能将survivin-asODN高效递送到肝癌SMMC-7221细胞,诱导人肝癌细胞的凋亡。更多还原

【Abstract】 Objective: To investigate the feasibility of using polyamidoamine(PAMAM)dendrimer as survivin antisense oligodeoxynucleotide(survivin-asODN) delivery system and to explore the effects of polyamidoamine dendrimer-survivin antisense oligodeoxynucleotide on the apoptosis of human hepatic cancer cell line SMMC-7721.Mehtods: The PAMAM-Survivin-asODN complex was prepared by mixing the 3.66 μg/L PAMAM dendrimer and 200 μg/L survivin antisense oligodeoxynucleotide.Meanwhile,the liposome-survivin-asODN complex was prepared as control.The shape and size of the complex were observed by transmission electron microscope and the zeta potential was measured by analytical tool.The encapsulating efficiency,DNA loading level,and the in vitro release speed were determined by ultraviolet spectrophotometer and centrifugation.The transfection efficiency of survivin-asODN in SMMC7721 cells was measured.The protein expression of survivin was measured by Western blotting analysis and the apoptosis rate of SMMC-7721 was assessed by flow cytometry(FCM).Results: The PAMAM-survivin-asODN complex was successfully prepared and the diameter of complex was shorter than that of liposome-survivin-asODN complex([64.9±9.60] vs [193.9±32.20],P<0.01).The zeta potential of the complex was higher than that of liposome-survivin-asODN complex([37.7±3.80] vs [22.6±2.20],P<0.05).No significant difference in the envelopment rates and loading levels were found between 2 groups.The in vitro release of survivin DNA lasted 14 days in PAMAM-survivin-asODN complex and 5 days in the liposome-survivin-asODN group.The transfection efficiency of PAMAM-survivin-asODN was higher than that ofliposome-survivin-asODN(P<0.05).The expression of survivin protein in PAMAM-survivin-asODN group was less than that of in the liposome-survivin-asODN group([26.8±5.65] vs [36.96±5.89],P<0.05) and the cell apoptosis rate was higher than that in the liposome-survivin-asODN group([60.3±8.25] vs [48.7±9.39],P<0.05).Conclusion: The polyamidoamine dendrimer can effectively deliver survivin-asODN into SMMC-7721 cells and induce SMMC-7721cells apoptosis.更多还原