聚酰胺树形分子-脂质体介导survivin反义寡核苷酸诱导肝癌细胞的凋亡

Survivin antisense oligonucleotide mediated by polyamidoaminedendrimer liposome induces apoptosis of hepatic cancer cells

目的:评价聚酰胺-胺型树枝状高聚合物(polyamidoamine,PAMAM)-脂质体复合物作为survivin反义寡核苷酸(survivin antisense oligonucleotide,survivin-asODN)载体传递系统的可行性,及其对人肝癌SMMC-7721细胞survivin表达、细胞凋亡的影响。方法:制备PAMAM与脂质体的复合物(PAMAM-脂质体),将survivin-asODN与PAMAM-脂质体或PAMAM混合,分别制备PAMAM-脂质体-survivin-asODN和PAMAM-survivin-asODN。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。将PAMAM-脂质体-survivin-asODN和PAM-AM-survivin-asODN转染SMMC-7721细胞,测定其转染率;Western blotting检测转染后SMMC-7721细胞中survivin蛋白的表达;流式细胞术检测SMMC-7721细胞的凋亡。结果:成功制备PAMAM-脂质体、PAMAM-脂质体-survivin-asODN和PAMAM-survivin-asODN。PAMAM-脂质体-survivin-asODN粒径与PAMAM-survivin-asODN粒径无显著差异[(189.33±15.42)vs(181.83±13.67)nm,P>0.05],包封率和载药率也无显著差异(P>0.05),但zeta电位高于后者[(42.83±7.14)vs(32.33±5.57)mV,P<0.05],PAMAM-脂质体-survivin-asODN转染SMMC-7721细胞的效率高于PAMAM-survivin-asODN[(73.33±9.29)%vs(60.67±7.81)%,P<0.05],转染后SMMC-7721细胞中survivin蛋白的表达较低(24.67±11.74 vs43.17±11.63,P<0.05),但细胞凋亡率高于PAMAM-survivin-asODN组SMMC-7721细胞[(73.31±12.59)%vs(52.67±12.19)%,P<0.05]。结论:PAMAM-脂质体能将survivin-asODN高效递送到人肝癌SMMC-7721细胞,诱导细胞凋亡。

Objective： To investigate the possibility of polyamidoaminedendrimer （PAMAM）-liposome for survivin antisense oligonucleotide （survivin-asODN） delivery system and explore the effects of PAMAM-liposome-survivin-asODN on survivin expression and apoptosis of human hepatic cancer cell line SMMC-7721. Methods： The liposome modified PAM- AM （PAMAM-liposome） was synthesized with liposome and PAMAM. Survivin-asODN was combined with the PAMAM- liposome or PAMAM to form PAMAM-liposome-survivin-asODN or PAMAM-survivin-asODN complexes. The shape and size of the two complexes were observed under a transmission electron microscope and their zeta potentials were measured with a zeta analytical tool. The encapsulating efficiency and DNA loading level were determined by ultraviolet spectrophotometer using a centrifuging method. PAMAM-liposome-survivin-asODN and PAMAM-survivin-asODN were transfected into SMMC-7721 cells, and the transfection efficiency was measured. The protein expression of survivin in SMMC-7721 cells was measured by Western blotting, and the apoptosis of SMMC-7721 cells was assessed by flow cytometry. Results： PAM- AM-liposome, PAMAM-liposome-survivin-asODN and PAMAM-survivin-asODN were successfully established. No signifi- cant difference appeared in diameter between PAMAM-liposome-survivin-asODN and PAMAM-survivin-asODN （ [ 189.33 ± 15.42 ] vs [ 181.83 ±13.671 nm, P 〉 0. 05）, as well as the encapsulating efficiency and drug loading level, but the zeta potential of PAMAM-liposome-survivin-asODN was higher than that of PAMAM-survivin-asODN （ [42.83 ±.14] vs [32.33 ±5.57] mV, P 〈0.05）. The transfection efficiency of PAMAM-liposome-survivin-asODN was higher than that of PAMAM-survivin-asODN （ [ 73.33 ±9.29） % vs [ 60.67 ±7.81 ] %, P 〈 0.05 ） in SMMC-7721 cells. The expression of survivin protein in SMMC-7721 cells of PAMAM-liposome-survivin-asODN group was less than that of PAMAM-survivin-asODN group （24.67 ±11.74 vs 43.17 ±11.63, P 〈 0.05 ） , while the apoptosis rate was higher than that of PAMAM-survivin-asODN （ [ 73.31 ±12.59 ] % vs [ 52.67 ±12.19 ] %, P 〈 0.05 ）. Conclusion： The PAM- AM-liposome can delivery survivin-asODN into 5MMC-7721 cells effectively and induce SMMC-7721 cell apoptosis.