聚酰胺树形分子高聚合物介导Survivin反义寡核苷酸诱导血管内皮细胞凋亡

Survivin antisense oligonucleotide mediated by polyamidoamine dendrimer induces apoptosis of vascular endothelial cells

目的:观察聚酰胺树形分子高聚合物作为survivin反义寡核苷酸(antisenseoligonucleotide,asODN)传递系统的可行性以及对人脐血管内皮细胞的survivin表达、细胞生长的影响。方法:实验于2005-05/2006-03在上海交通大学微纳米科学技术研究院微纳制造技术国家重点实验室完成。①将200μg/Lsurviving-asODN和3.66μg/L聚酰胺制备聚酰胺反义基因复合物,同时制备阳离子脂质体反义基因复合物作为对照。②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,离心法和紫外分光分度仪测定复合物的包封率、载药率和体外DNA释放速度。③将上述两种基因载体复合物转染人脐血管内皮细胞,测定其转染效率;Westernblotting检测转染后细胞中survivin蛋白的表达;流式细胞术检测两组细胞的凋亡率。结果:①成功制备聚酰胺反义基因载体系统聚酰胺-survivin-asODN。该复合物的粒径小于脂质体-survivin-asODN复合物(P<0.01),但zeta电位高于后者(P<0.05);两者基因载药率、包封率无显著差异(P>0.05);聚酰胺对DNA持续释放14d,但脂质体只持续5d。②聚酰胺-survivin-asODN转染人脐血管内皮细胞的效果强于脂质体-survivin-asODN(P<0.05),转染后人脐血管内皮细胞siurvivin蛋白的表达低于脂质体复合物(P<0.05),该细胞的凋亡率高于脂质体复合物(P<0.05)。结论:聚酰胺能将Survivin-asODN高效递送到人脐血管内皮细胞,降低survivin蛋白的表达并诱导血管内皮细胞凋亡。

AIM： To investigate the possibility of polyamidoamine （PAMAM） dendrimer as survivin antisense oligonucleotide （ASODN） delivery system and explore the effects of PAMAM dendrimer-survivin ASODN on the survivin expression and the growth of human umbilical vascular endothelial cells （HUVECs）. METHODS： The experiment was carried out in the National Key Laboratory of Nano/Micro Fabrication Technology, Research Institute of Micro/nanometer Science ＆ Technology, Shanghai Jiao Tong University from May 2005 to March 2006.① PAMAM anti-sense gene transfection complex was generated by mixing 3.66 μg/L PAMAM dendrimer and 200μg/L survivin ASODN, while the liposome anti-sense gene transfection complex was manufactured as controls.②The 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 release progress in vitro of the compound were determined by ultraviolet spectrophotometer in centrifuging method.③The transfection efficiency of survivin-ASODN in HUVECs was measured. The protein expression of survivin was measured by Western blotting analysis, and the apoptosis rate of HUVECs was assessed by flow cytometry. RESULTS： ①The PAMAM-survivin-ASODN complex was successfully prepared and its diameter was less than that of liposome-survivin-ASODN complex （P 〈 0.01）, but the zeta potential was higher than that of liposome-survivin-ASODN complex （P 〈 0.05）. There was no significant difference between two groups for envelopment ratio and loaded rate （P 〉 0.05）. The PAMAM released for 14 days and the liposome only for 5 days. ② The transfection efficiency for PAMAM-survivin-ASODN was higher than that for liposome-survivin-ASODN （P 〈 0.05）. The expression of survivin protein for PAMAM-survivin-ASODN was less than that for liposome-survivin-ASODN （P 〈 0.05）, and the cell apoptosis rate was higher than that for liposome-survivin-ASODN （P 〈 0.05）. CONCLUSION： The PAMAM dendrimer can delivery survivin-ASODN into HUVECs effectively, reduce the expression of survivin and induce HUVEC apoptosis.