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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 study the dendritic cell (DC) tumor vaccine loaded with hTERT gene fragment and its anti-tumor effect. Methods: The hTERT gene fragment was constructed and lentiviral vector was constructed. The expression of hTERT gene in Lenti-hTERT transduced 293T cells was detected by immunocytochemistry. DCs derived from cord blood were isolated and identified by phase-contrast microscopy and flow cytometry. DC vaccine was prepared by Lenti-hTERT transduction of DC vaccine. DC vaccine stimulated allogeneic T lymphocytes to induce specific CTLs and their proliferation and specific cytotoxicity against target cells were detected by MTT assay. Results: Lenti-hTERT with hTERT gene fragment was successfully constructed and the titer of virus was 5.88 × 106 U / mL. The expression of hTERT in transduced cells was significantly increased for> 2 months. The lentiviral transduction became the DC vaccine loaded with hTERT, and the ability of DCs loaded with hTERT antigen to stimulate the proliferation of lymphocytes was significantly better than those without hTERT (P <0.01). The cytotoxicity of DCs induced by DCs loaded with hTERT antigen on HepG2 hepatoma cells was significantly higher than that induced by unmodified DCs (P <0.01), and the tumor inhibition rates were 54.78% and 12.08%, respectively. CTLT of hTERT negative 293T cells And CTLT were lower and the difference was not statistically significant, P> 0.05. CONCLUSION: The DC vaccine prepared by lentivirus-mediated hTERT modification has an enhanced ability to stimulate the proliferation of T cells. The induced CTL has a specific and significantly enhanced killing effect on telomerase-positive HepG2 tumor cells.