目的:探讨利用小鼠α1,3半乳糖基转移酶基因进行肝癌基因治疗的可行性.方法:将pcDNA3.1-α1,3GT真核表达载体以脂质体介导法转染人肝癌细胞HuH7,经G418压力筛选出稳定表达的阳性克隆,经生长曲线法,平板克隆形成试验,流式细胞仪等试验分析稳定表达株相关生物学特性变化.结果:转染有α1,3GT的HuH7经RT-PCR法检测到有α1,3GT的表达,免疫荧光镜检测其细胞表面有明显的Galα(1,3)Gal糖表位表达,转染细胞的增殖能力明显降低,同时促进细胞凋亡.结论:转染α1,3-半乳糖基转移酶基因可明显抑制人肝癌细胞HuH7的恶性生物学行为,具有潜在的临床应用价值.%Objective : To explore the feasibility of gene therapy by the murine α1 ,3 - galactosyltransferase in hepatocellular carcinoma. Methods : The euarokaytic vector pCDNA - α1 ,3GT containing the full length cDNA of α1 3 - galactosyltransferase gene was transfected into the HuH7 cell line by LipofectaminerTM 2000. Then stable expression clones were selected with G418 and appraised by RT - PCR and immunofiuorescence . The growth and proliferative capacity were analyzed by making cell growth curves and the colony formation assay , respectively. The apoptosis and cell cycles were detected by using flow cytometry. Results: Obtained the positive clones by screening with G418 for 3 - 4 weeks, compared with the cells which having been transfected with pure plasmid ( HuH7 - pcDNA ) and those not having been transfected ( HuH7 ) , the expression level of α1 ,3GT gene mRNA and α1 ,3 Gal evidently was found in the cells which having beentransfected with pcDNA - α1 , 3 GT plasmid ( HuH7 - αGT ) . The proliferativecapacity of transfected cells was demonstrated to be inhibited by curve of cell growth and the apoptotic rate was ( 5. 70 ±0. 10 ) ％ , rising up obviously( P ＜0. 05 ) . The results of the colony formation assay showed that no significant differences in the rate of colony formation of HuH7 - αGT compared to HuH7 - pcDNA( P = 0. 238 ). Conclusion: Transfection of the α1 , 3GT could obviously inhibit the malignancy of HuH7 cells.
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