特异性杀伤肝癌细胞的噬菌体药物载体的构建

目的以T4噬菌体为载体,构建特异性杀伤肝癌细胞的噬菌体药物载体,并对其选择性细胞毒活性进行初步评价。方法通过噬菌体文库筛选肝癌细胞特异性结合肽(tumor specific peptide,TSP),利用分子克隆技术和噬菌体体外展示技术,将其与噬菌体小衣壳蛋白Soc重组,并展示在T4噬菌体表面。同时通过1-乙基(-3-二甲基氨基丙级基)碳酰二亚胺[1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride,EDC]化学法,将表阿霉素(epido-xorubicin,Epi)偶联在噬菌体衣壳表面,采用MTT法在细胞水平上检测噬菌体药物载体对人肝癌细胞Bel-7402和正常肝细胞HL-7702的选择性杀伤作用。结果 Soc-TSP融合蛋白以846拷贝/噬菌体颗粒的密度展示在T4噬菌体表面,Epi载药率达每个噬菌体颗粒表面近2 000个药物分子。与游离的Epi相比,构建的T4噬菌体药物载体对肝癌Bel-7402细胞的杀伤作用提高了4⁶倍,而对正常肝细胞HL-7702的影响不大。结论肿瘤特异噬菌体药物载体可提高药物对肿瘤细胞的靶向性以及肿瘤细胞对药物分子的摄入,从而实现对肿瘤细胞的特异性杀伤。

Construction of bacteriophage as a carrier of drug specific for hepatocellular carcinoma cells

Objective To construct the bacteriophage as a carrier of drug specific for hepatocellular carcinoma(HCC) cells,and preliminarily evaluate its selective cytotoxicity.Methods Tumor specific peptide(TSP) were screened from phage-displayed random peptide library,recombined to bactriophage small capsid protein Soc by molecular cloning and an in vitro assembly strategies,and displayed on the surface of T4 bacteriophage.Epidoxorubicin(Epi)was loaded onto the bacteriophage capsid by using 1-ethyl-3(-3-dimethylaminopropyl)carbodiimide hydrochloride(EDC).In vitro tumor selective cytotoxicity of the constructed drug-carrying bacteriophage particles to HCC Bel-7402 cells and normal liver HL7702 cells was evaluated by MTT method at cell level.Results Soc-TSP fusion protein was displayed on the surface of T4 bacteriophage at a density of 846 copies/particle.The Epi-loading rate was about 2 000 molecules/particle.The killing rate of T4-carried Epi to Bel-7402 cells was 4 - 6 times higher than that of free Epi.However,T4-carried Epi showed no significant effect on HL-7702 cells.Conclusion Tumor-selective bacteriophage particle exerted their tumorkilling property via promoting the targeted drug delivery as well as the drug uptake by the tumor cells.