脂质-聚阳离子-CDKN1B质粒复合物的构建、特性和转染

摘要

Objective To develop an efficient non-viral gene delivery system in order to transfer CDKN1B gene efficiently into lung and liver carcinoma cells. Methods A recombinant plasmid composed of CDKN1B sequence and EYFP as reporter gene was constructed and identified. The recombinant DNA was then formulated the lipids-polycation- DNA complexes(LPDs) with protamine sulfate. Several kinds of lung and liver carcinoma cells were transfected by means of LPDs. The physicochemical properties of LPDs were investigated using PCS method and TEM, respectively. The expression of EYFP in A549 cells was observed under fluorescent microscope and evaluated by flow cytometry analysis. Finally, the production of CDKN1B protein in transfected LLC, Chang and 7721 cells was identified by Western blot analysis. Results The average diameter of the LPDs were 167 nm with the polydispersity index of 0.35. The average zeta potential of LPDs was +32.6 mV. LPDs look like a sunken sphere. The fluoresent microscope picture clearly indicated the expression of EYFP in A549 cells. The flow cytometry result showed that the transfection efficiency of LPDs in A549 cells was comparable with that of LipofectAMINE(R), the positive control. Western blot analysis confirmed the production of CDKN1B protein in LLC, Chang and 7721 cells transfected with LPDs, while no CDKN1B protein was detected in cells transfected with naked DNA. Conclusion The construction of the recombinant plasmid is successful. LPDs can deliver the recombinant plasmid to lung carcinoma cells and liver carcinoma cells with high efficiency. Therefore, this kind of gene delivery system has the potential uses for the treatment of lung and liver cancer.%目的研究开发一种能有效转染 CDKN1B基因进入肺和肝癌细胞的非病毒基因传递系统.方法重组构建了含有 CDKN1B序列和EYFP报告基因的质粒.该重组DNA与鱼精蛋白缩合后再与脂质体作用形成脂质-聚阳离子-DNA复合物(lipids-polycation-DNA-complex, LPDs),分别用激光粒度仪法和透射电镜法研究该LPDs的理化性质;用该LPDs转染几种肺癌、肝癌细胞;该LPDs中的EYFP在A549 细胞中的表达情况用荧光显微镜和流式细胞仪观察和评价;在LLC肺癌细胞、Chang正常肝细胞和7721肝癌细胞中表达的CDKN1B蛋白用Western印迹方法分析.结果该LPDs为凹陷的球状;平均粒径是167 nm,多分散指数为0.35;平均zeta电位为+32.6 mV;从A549细胞的荧光显微照片可清楚观察到EYFP的表达;A549细胞的流式细胞仪分析结果显示,LPDs的转染效率与阳性对照LipofectAMINE(R) 的转染效率相当;Western印迹法分析显示,以LPDs装载的 CDKN1B质粒在LLC细胞、常细胞和7721细胞中表达CDKN1B蛋白,而裸 CDKN1B质粒没有表达.结论脂质-聚阳离子- CDKN1B质粒复合物的构建是成功的.LPDs能够将 CDKN1B质粒传递到肺癌细胞和肝癌细胞,并获得高效率的表达.因此,这种基因传递系统有望开发用作肺癌和肝癌的基因治疗传递系统.