Zhang Ling, Sun Xun, Zhang Hong-wei, Sang Ying-ying, Yi Tao, Qiao Shou-yi
School of Life Sciences, Fudan University, Shanghai, PR China.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi. 2005 Oct;22(5):502-6.
To develop an efficient non-viral gene delivery system in order to transfer CDKN1B gene efficiently into lung and liver carcinoma cells.
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.
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, 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.
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.
开发一种高效的非病毒基因递送系统，以便将CDKN1B基因有效地转入肺癌和肝癌细胞。
构建并鉴定由CDKN1B序列和作为报告基因的EYFP组成的重组质粒。然后将重组DNA与硫酸鱼精蛋白配制形成脂质-聚阳离子- DNA复合物（LPDs）。通过LPDs转染几种肺癌和肝癌细胞。分别采用电位分析法（PCS）和透射电子显微镜（TEM）研究LPDs的理化性质。在荧光显微镜下观察A549细胞中EYFP的表达，并通过流式细胞术分析进行评估。最后，通过蛋白质免疫印迹分析鉴定转染的LLC、Chang和7721细胞中CDKN1B蛋白的产生。
LPDs的平均直径为167 nm，多分散指数为0.35。LPDs的平均ζ电位为+32.6 mV。LPDs呈凹陷球体状。荧光显微镜照片清楚地显示了A549细胞中EYFP的表达。流式细胞术结果表明，LPDs在A549细胞中的转染效率与阳性对照LipofectAMINE相当。蛋白质免疫印迹分析证实，用LPDs转染的LLC、Chang和7721细胞中产生了CDKN1B蛋白，而用裸DNA转染的细胞中未检测到CDKN1B蛋白。
重组质粒构建成功。LPDs可将重组质粒高效递送至肺癌细胞和肝癌细胞。因此，这种基因递送系统在肺癌和肝癌治疗方面具有潜在应用价值。
