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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 evaluate the feasibility of using polyamide-amine polyamidoamine (PAMAM)-liposomal complexes as survivin antisense oligonucleotide (survivin-asODN) vector delivery system. Effects of survivin expression and apoptosis in human hepatocellular carcinoma SMMC-7721 cells. Methods: PAMAM and liposome complexes (PAMAM-liposomes) were prepared. Survivin-asODN was mixed with PAMAM-liposomes or PAMAM to prepare PAMAM-liposomes-survivin-asODN and PAMAM-survivin-asODN, respectively. . Transmission electron microscopy was used to observe the morphology and particle size of the complexes. The zeta potential of the complexes was measured by zeta potential analyzer. The encapsulation efficiency and drug loading rate of the complexes were determined by centrifugation and ultraviolet spectrophotometer. PAMAM-liposomal-survivin-asODN and PAM-AM-survivin-asODN were transfected into SMMC-7721 cells and the transfection efficiency was measured. Western blotting was used to detect the expression of survivin protein in SMMC-7721 cells after transfection; The apoptosis of SMMC-7721 cells was detected. RESULTS: PAMAM-liposomes, PAMAM-liposomal-survivin-asODN and PAMAM-survivin-asODN were successfully prepared. The particle size of PAMAM-liposomal-survivin-asODN was not significantly different from that of PAMAM-survivin-asODN [(189.33±15.42) vs (181.83±13.67) nm, P>0.05], and the entrapment efficiency and drug loading rate were not Significant difference (P>0.05), but the zeta potential was higher than that of the latter [(42.83±7.14) vs (32.33±5.57) mV, P<0.05], and the PAMAM-liposomal-survivin-asODN transfected SMMC-7721 cells. The efficiency was higher than that of PAMAM-survivin-asODN [(73.33±9.29)% vs (60.67±7.81)%, P<0.05], and the expression of survivin protein was lower in SMMC-7721 cells after transfection (24.67±11.74 vs43.17± 11.63, P<0.05), but the rate of apoptosis was higher in SMMC-7721 cells than in PAMAM-survivin-asODN group [(73.31±12.59)% vs (52.67±12.19)%, P<0.05]. CONCLUSION: PAMAM-liposomes efficiently deliver survivin-asODN to human hepatocellular carcinoma SMMC-7721 cells and induce apoptosis.