肝细胞癌基因治疗的体外实验研究

Study on Gene Therapy of Hepatocellular Carcinoma in Vitro

目的 :构建一种对肝细胞癌有直接、间接治疗作用的重组腺病毒。方法 :用PCR方法扩增CD基因和glyES基因片段 ,插入腺病毒穿梭质粒。用细菌内同源重组方法与腺病毒骨架质粒重组构建出重组腺病毒质粒。经脂质体介导转染293包装细胞扩增获取重组腺病毒rAd CDglyES。用MTT法检测其对肝癌细胞株 (SMMC -7721)的生长抑制率及其表达产物对经ECGF处理的脐静脉血管内皮细胞 (ECV -304)增殖的影响。结果 :纯化的重组腺病毒滴度为1×1013 3TCID50/L ,对SMMC 7721的生长抑制率达 (82 1±8 3) % ,与对照组 (24 6±14 1) %比较有显著性差异 (P<0 01)。浓缩的转染了该重组腺病毒的细胞培养上清液对ECV 304细胞增殖的抑制率为(78 7±1 8) % ,而同样浓缩的转染了对照重组腺病毒rAd CD的细胞培养上清液的抑制率仅为 (24 2±9 7) % ,两者亦有显著性差异 (P<0 01)。结论 :rAd

Objective:To construct a recombinant adenovirus which is capable of both direct and indirect treatment to hepatocellular carcinoma.Methods:CD and glyES genes were respectively amplified by PCR,and inserted into the shuttle plasmid of adenovirus.A method of homologous recombination in bacteria was used to construct prAdˉCdglyES.The recombination adenovirus was transfected to293cells by liposome,in which rAdˉCDglyES was packaged and generated.MTT method was used to detect the growth inhibition effect of rAdˉCDglyES on SMMCˉ7721and the growth inhibition effect of expressing products on ECVˉ304.Results:The tite of rAdˉCdglyES was1×10 13.3 TCID 50 /L,the inhibiting rate on SMMCˉ7721cells was(82.1±8.3)%,and had a significant difference compared with control which was(24.6±14.1)%(P<0.01).The inhibiting rate of ECVˉ304cells from concentrated cellular culture supernatant was(78.7±1.8)%,and was significant difference compared with control which was(24.2±9.7)%(P<0.01).Conclusion:rAdˉCDglyES is capable of both direct and indirect growth inhibition effect on hepatocellular carcinoma in vitro.