负载hTERT基因片段的DC肿瘤疫苗制备及其抗肿瘤作用的研究

Anti-tumor effects and construction of lentivirus-hTERT mediated DC vaccine

目的:研究负载hTERT基因片段的树突状细胞(DC)肿瘤疫苗及其抗肿瘤作用。方法:构建负载hTERT基因片段的慢病毒载体,采用细胞免疫化学技术检测Lenti-hTERT转导的293T细胞中hTERT基因的表达;分离制备脐血来源的DC,通过相差显微镜和流式细胞仪鉴定;Lenti-hTERT转导DC细胞制备DC疫苗;DC疫苗刺激同种T淋巴细胞诱导特异性CTL并用MTT法检测其增殖能力和对靶细胞的特异性杀伤能力。结果:成功构建负载hTERT基因片段的Lenti-hTERT,病毒滴度达5.88×106U/mL;转导后细胞中hTERT的表达显著升高,持续时间>2个月;分离制备脐血来源的DC,经慢病毒转导成为负载hTERT的DC疫苗,负载了hTERT抗原的DC刺激淋巴细胞增殖的能力明显优于未负载hTERT的DC(P<0.01),增殖指数均>1.5。负载了hTERT抗原的DC诱导CTLT对HepG2肝癌细胞的杀伤能力明显高于未经修饰的DC所诱导CTLN(P<0.01),抑瘤率分别为54.78%和12.08%;对hTERT阴性的293T细胞CTLN和CTLT的抑制率均较低且差异无统计学意义,P>0.05。结论:用慢病毒介导hTERT修饰制备的DC疫苗,其刺激T细胞增殖的能力增强,诱导的CTL对端粒酶阳性的HepG2肿瘤细胞具有特异而且显著增强的杀伤效应。

OBJECTIVE：To produce gene modified DC vaccine transduced with lentiviral vector encoding human hTERT and to investigate its anti-tumor effect.METHODS：Recombinant vector Lenti-hTERT was constructed and hTERT expression in transduced cells was detected with immunocytochemical staining.Dendritic cells from cord blood were isolated and induced,and their morphology and phenotype were analyzed by flow cytometer.Dendritic cells were transduced with Lenti-hTERT to get gene modified DC vaccine.The proliferation of allogeneic T lymphocytes stimulated by and the cytotoxincity of CTL activated by these DCs were detected with MTT assay.RESULTS：The recombinant vector Lenti-hTERT was successfully constructed.The titration of lenti-hTERT was 5.88×106 U/mL.hTERT expressed highly in the cells transduced with lenti-hTERT and last for more than two months.Modified DC vaccine Lenti-hTERT DC was prepared.The stimulatory capacity of the Lenti-hTERT DC in the allogeneic T lymphocytes reaction was markedly enhanced as compared with the DC control group （P0.01）.The stimulator indexes （SI） were all above 1.5 in the Lenti-hTERT DC group.The killing activity of CTL stimulated with Lenti-hTERT-DC （CTLT） to HepG2 was significantly higher than that of CTL stimulated with DC （CTLN） （P0.01）,and the killing rates were 54.78% and 12.08% respectiveies.But to 293T,both CTLT and CTLN had low killing activity （P0.05）.CONCLUSION：The gene modified DC vaccine transduced with lenti-hTERT can stimulate the proliferation of allogeneic T lymphocytes more effectively,and induce CTL to kill hTERT＋ HepG2 specificly.