背景:传统的病毒载体及非病毒载体在基因治疗中均存在明显的缺点,采用纳米材料作为反义寡核苷酸载体有望获得更好更安全的基因转导,提高基因治疗的有效性和安全性。实验拟采用聚酰胺纳米分子介导反义寡核苷酸阻断survivin表达,诱导大肠癌凋亡。目的:探讨聚酰胺树形分子高聚合物介导survivin反义寡核苷酸(survivin antisense oligonucleotide,survivin-asODN)转染结直肠癌SW620细胞的可行性以及对结直肠癌SW620细胞survivin表达、细胞凋亡的影响。设计、时间及地点:肿瘤基因治疗体外实验,于2007-09/2008-05在上海交通大学微纳米科学技术研究院生物纳米工程研究室及南方医科大学珠江医院中心实验室完成。材料:人结直肠癌细胞株SW620购自中国科学院上海细胞研究所,聚酰胺树形分子由上海交通大学微纳米研究院生物纳米工程研究室崔大祥教授提供,转染试剂脂质体LipofectamineTM2000购自美国Invitrogen公司。survivin-asODN由上海生工公司合成。方法:采用300μg/L survivin-asODN和4.06μg/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。透射电镜观察复合物的形态,激光散射粒径分析仪测定粒径,zeta电位分析仪测定zeta电位,离心法和紫外分光光度仪测定复合物的的包封率、载药率和体外DNA释放速度。将上述两种基因转染复合物转染结直肠癌细胞,测定其转染效率;Western blotting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。主要观察指标:阳离子脂质体-survivin-asODN复合物及聚酰胺-survivin-asODN复合物的粒经,zeta电位,基因载药率,包封率,释放率,转染后survivin表达率及对凋亡的诱导率。结果:聚酰胺-survivin-asODN复合物的粒径小于脂质体-survivin-asODN复合物的粒径(P<0.01),但zeta电位高于后者(P<0.05);基因载药率、包封率两组差异无显著性意义;聚酰胺对DNA持续释放14d,但脂质体只持续5d。聚酰胺-survivin-asODN转染结直肠癌细胞的效果强于脂质体-survivin-asODN(P<0.05)。转染后结直肠癌细胞survivin蛋白的表达低于脂质体复合物(P<0.05),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高效递送到结直肠癌SW620细胞,降低survivin蛋白表达并诱导结直肠癌细胞凋亡。 BACKGROUND: Traditional viral vectors and non-viral vectors have obvious shortcomings in gene therapy. It is expected that the use of nanomaterials as antisense oligonucleotide vectors will lead to better and safer gene transduction and enhance the effectiveness and safety of gene therapy Sex. Antisense oligonucleotides are used to block the expression of survivin and induce apoptosis in colorectal cancer. OBJECTIVE: To investigate the feasibility of transfection of survivin antisense oligonucleotide (survivin-asODN) into colorectal cancer SW620 cells mediated by PAMAM as well as its effect on the expression of survivin in SW620 colorectal cancer cells, Effect of apoptosis. DESIGN, TIME AND SETTING: In vitro experiments on tumor gene therapy were performed at the Laboratory of Bio-Nano Engineering, Institute of Micro and Nano Science and Technology, Shanghai Jiaotong University, and Central Laboratory of Zhujiang Hospital, Southern Medical University from September 2007 to May 2008. MATERIALS: Human colorectal cancer cell line SW620 was purchased from Shanghai Institute of Cell Research, Chinese Academy of Sciences. Polyamide dendrimer was provided by Prof. Cui Daxiang, Laboratory of Bio-Nano-Engineering, Institute of Micronanobiology, Shanghai Jiaotong University. Transfection reagent LipofectamineTM2000 was purchased from USA Invitrogen Corporation. Survivin-asODN synthesized by Shanghai Shengongsi. Methods: Polyamide antisense gene complex was prepared by using 300 μg / L survivin-asODN and 4.06 μg / L polyamide, and the cationic liposome antisense gene complex was prepared as a control. The morphology of the composite was observed by transmission electron microscopy. The particle size was measured by laser scattering particle size analyzer. The zeta potential was measured by zeta potential analyzer. The entrapment efficiency, drug loading rate and DNA release rate in vitro were determined by centrifugation and ultraviolet spectrophotometry . Transfection of the above two gene transfection complexes into colorectal cancer cells was performed to determine the transfection efficiency. The expression of survivin protein was detected by Western blotting. The apoptosis rates of the two groups were detected by flow cytometry. MAIN OUTCOME MEASURES: Granulosis, zeta potential, gene drug-loading rate, entrapment efficiency and release rate of cationic liposomes-survivin-asODN complex and polyamide-survivin-asODN complex, the expression of survivin after transfection Induction of apoptosis. Results: The particle size of the polyamide-swurvivin-asODN complex was smaller than that of the liposome-survivin-asODN complex (P <0.01), but the zeta potential was higher than that of the latter (P <0.05) There was no significant difference in the encapsulation efficiency between the two groups. Polyamide sustained DNA release for 14 days, but the liposome only lasted for 5 days. Polyamide-survivin-asODN transfected colorectal cancer cells were more effective than liposome-survivin-asODN (P <0.05). The expression of survivin protein in colorectal cancer cells after transfection was lower than that in liposome complex (P <0.05), and the apoptosis rate was higher than that in liposome complex (P <0.05). CONCLUSION: Polyamide can efficiently deliver survivin-asODN to colorectal cancer SW620 cells, reduce survivin protein expression and induce apoptosis of colorectal cancer cells.