磁性小干扰RNA纳米微粒的制备及其转染人膀胱癌细胞对沉默survivin基因表达影响的研究(英文)

Construction of magnetic small interfering RNA nanoparticles and effects of its transfection on silencing survivin gene expression and inducing human bladder cancer cell apoptosis

目的研究磁性小干扰RNA(siRNA)重组质粒纳米微粒的制备及其在外磁场协同作用下转染人膀胱癌细胞对膀胱癌细胞凋亡和沉默survivin基因表达的影响。方法利用化学共沉淀法制备葡聚糖磁性纳米微粒(DMN)并作为基因载体,通过多聚赖氨酸静电吸附作用制备磁性siRNA-survivin-DMN重组质粒纳米微粒,并在外磁场的协同作用下体外转染人膀胱癌BIU-87细胞,分别采用四甲基偶氮唑蓝比色法、末端脱氧核苷酸转移酶介导的dUTP缺口末端标记技术法、半定量逆转录聚合酶链反应和Western Blotting检测BI-U-87细胞的生长抑制率(IR)、凋亡指数(AI)、survivin mRNA及其蛋白的相对表达水平。结果构建成功的siRNA-DMN直径、有效粒径、比饱和磁化强度分别为10～12nm、94.8nm、0.19emu/g。DMN在外磁场的协同作用下转染siRNA重组质粒入BIU-87细胞后的IR(39.60%)、AI(28.72%)均分别显著高于对照组和无磁场协同作用的siRNA-DMN组(1.99%、3.75%和20.96%、16.71%)(均P<0.05),survivin mRNA及其蛋白相对表达水平均显著低于后2组。结论磁性siRNA-DMN纳米微粒在外磁场的协同作用下转染BIU-87细胞后可有效抑制survivin基因表达并诱导细胞凋亡,为膀胱肿瘤的磁靶向基因治疗提供实验依据。

[Objective] To investigate the construction of the magnetic small interfering RNA （siRNA） plasmid nanoparticles and the effects of its transfection on silencing survivin gene expression and inducing human bladder cancer cells apoptosis in combination with external magnetic fields in vivo. [Methods] The siRNA-survivin recombinant plasmid specific targeted survivin was synthesized in previous experiment. DMN were prepared by chemical coprecipitation method and used as a gene carrier. The magnetic siRNA-survivin-DMN were constructed by static electricity of polylysine and transferred into human bladder cancer BIU-87 ceils with the help of external magnetic fields. The growth inhibitory rate （IR） % of BIU-87 cells was observed by 3-[4, 5-dimethylthiazol-2-yl]-2, 5- diphenyl tetrazolium bromide＇s test and the apoptosis index （A1） % was detected by transferase-mediated dUTP nick end labeling method. The relatively transcription levels of survivin mRNA and protein expression were respectively detected by semi-quantitive reverse transcription polymerase chain reaction and Western Blotting techniques. [Resuits] The diameter, effective diameter and saturation magnetization of magnetic siRNA-DMN were about 10-12 nm, 94.8 nm and 0.19 emu/g, respectively. The IR （39.60%） and AI （28.72%） in BIU-87 cells treated by siRNA- DMN in combination with external magnetic fields were significantly higher than those in the control group and single siRNA-DMN group, respectively （P 〈0.05）, while the relative expression of survivin mRNA and protein was sig- nificantly lower （P 〈0.05） [Conclusion] The magnetic siRNA-DMN plasmid nanoparticles could effectively inhibit survivin expression and induce BIU-87 cells apoptosis after transferred into ceils in combination with external magnetic fields which provided experimental basis for the magnetic targeting gene therapy of bladder tumor.