摘要
目的构建一种具有靶向性的非病毒基因治疗载体高尔基体蛋白73(GP73)-脂质体lipoplexes,并检测其对肝癌细胞Hep G2和正常肝细胞HL-7702的转染效率和凋亡的影响。方法以水溶性碳二亚胺为交联剂将GP73与lipoplexes偶联。用GP73-lipoplexes运载标记有绿色荧光蛋白(green fluorescent protein,GFP)荧光报告基因的PGenesil-l质粒转染肝癌细胞组Hep G2和肝正常细胞组HL-7702,通过荧光显微镜观察被转染细胞GFP的表达情况,流式细胞术检测转染效率。并用GP73-lipoplexes运载含治疗基因HSVtk的质粒PBI-SUR-TK转染肝癌细胞和肝细胞,经RT-PCR和Western blot检测细胞中HSVtk表达情况,流式细胞术和CCK-8检测细胞凋亡情况。结果经GP73修饰的lipoplexes转染的肝癌细胞组[(26.37±0.76)%]比肝细胞组[(9.68±0.57)%]有更高的转染效率和更多的HSVtk基因的表达,肝癌细胞增殖能力明显减弱,凋亡显著。结论 GP73修饰的lipoplexes介导的HSVtk自杀基因系统对肝癌细胞有治疗作用,可以尝试作为一种靶向性杀伤肝癌细胞的非病毒基因转运载体。
Objective To construct a non-viral gene therapy vector(GP73 modified lipoplexes) and to assess its delivery efficiency and impact on apoptosis in hepatoma cell line Hep G2 and normal liver cell line HL-7702. Methods Lipoplexes were conjugated with human GP73 using water-soluble carbodidimide, a crossing agent. Then, GP73-lipoplexes were used to carry p Genesil-l plasmid containing EGFP gene and transfect hepatoma cell line Hep G2 and normal liver cell line HL-7702. Fluorescence microscopy and flow cytometry were used to assess the transfection efficiency. GP73-lipoplexes were then used to carry PBI-SUR-TK plasmid containing the therapeutic gene HSVtk and transfect hepatoma cells and normal hepatocytes. RT-PCR and Western blot were used to detect the expression of HSVtk in cells, and flow cytometry and CCK-8 assay were used to detect apoptosis. Results Compared with HL-7702 cells, Hep G2 cells had higher transfection efficiency [(9.68±0.57)% vs(26.37±0.76)%], obvious HSVtk gene production, and significant apoptosis. Conclusion GP73 modified lipoplexes can be an effective tool to improve the transfection efficiency and therapeutic effects of exogenous genes in hepatoma cells.
作者
孟凡秀
张琪
姚志坚
袁洋洋
曾思衡
刘畅
李勇芳
张英敏
赵娜
陆嘉慧
张智宣
李高凯
于保锋
郭睿
王海龙
解军
李高鹏
徐钧
Meng Fanxiu;Zhang Qi;Yao Zhijian;Yuang Yangyang;Zeng Siheng;Liu Chang;Li Yongfang;Zhang Yingmin;Zhao Na;Lu Jiahui;Zhang Zhixuan;Li Gaokai;Yu Baofeng;Guo Rui;Wang Hailong;Xie Jun;Li Gaopeng;Xu Jun(Department of Biochemistry and Molecular Biology,Shanxi Medical University,Taiyuan 030001,China;College of Dental Medicine,Shanxi Medical University,Taiyuan 030001,China;School of Public Health,Shanxi Medical University,Taiyuan 030001,China;Department of General Surgery,Affliated Tumor Hospital of Shanxi Medical University,Taiyuan 030013,China)
出处
《中华临床医师杂志(电子版)》
CAS
2017年第19期2273-2279,共7页
Chinese Journal of Clinicians(Electronic Edition)
基金
国家自然科学基金(30901821
81172136)
山西省自然科学基金(2015011113)