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目的构建表达人类白细胞抗原-E(HLA-E)基因慢病毒载体,探讨慢病毒介导HLA-E基因在肿瘤免疫中的意义。方法2006年10月至2007年11月在中山大学附属第三医院肝移植中心应用pGC-E1-GFP-HLA-E慢病毒载体转导肝癌HepG2细胞,检测慢病毒载体的转导效率、细胞内HLA-E信使核糖核酸(mRNA)及蛋白质水平的表达。结果pGC-E1-GFP-HLA-E慢病毒载体转导肝癌HepG2细胞72h的转导效率为(95.0±1.3)%;通过RT-PCR检测24h后细胞内HLA-E mRNA水平是肝癌HepG2细胞的(8.73±1.05)倍,72h后为(293.48±42.01)倍,差异具有统计学意义(P<0.01)。细胞免疫组化显示转导HLA-E后细胞内的蛋白水平明显增强。结论慢病毒载体系统能够使转导的基因在靶细胞中得到稳定表达,是基因治疗中的理想载体。
Objective To construct a lentiviral vector expressing human leukocyte antigen-E (HLA-E) gene and investigate the significance of lentivirus-mediated HLA-E gene in tumor immunity. Methods From October 2006 to November 2007, hepatocellular carcinoma HepG2 cells were transduced with pGC-E1-GFP-HLA-E lentiviral vector at the Third Affiliated Hospital of Sun Yat-sen University Liver Transplant Center to detect the lentiviral vector transduction efficiency, intracellular HLA-E messenger ribonucleic acid (mRNA) and protein expression. Results The transduction efficiency of HepG2 cells transfected with pGC-E1-GFP-HLA-E lentiviral vector for 72 h was (95.0 ± 1.3)%. The mRNA level of HLA-E in HepG2 cells was detected by RT- (8.73 ± 1.05) times and 72.4 hours (293.48 ± 42.01) times, the difference was statistically significant (P <0.01). Immunohistochemistry showed that the level of intracellular protein was significantly increased after transduction of HLA-E. Conclusion The lentiviral vector system can make the transduced gene stably expressed in target cells and is an ideal vector in gene therapy.