【摘要】 目的:构建pEGFP-C3-TCR BV12-3表达载体并初步研究其抗肿瘤作用。方法:TCR BV12-3基因片段从pGEM-T-TCR BV12-3载体上酶切并克隆至pEGFP-C3载体中,通过脂质体将pEGFP-C3-TCR BV12-3表达载体转染外周血单个核细胞(PBMCs),48小时后荧光显微镜观察转染效率。PBMCs细胞、pEGFP-C3载体转染的PBMCs细胞、pEGFP-C3-TCR BV12-3表达载体转染的PBMCs细胞分别与肝癌细胞BEL-7402和宫颈癌细胞HeLa共培养24 h,显微镜观察肿瘤细胞的生长情况。结果:测序证实TCR BV12-3基因片段成功亚克隆至pEGFP-C3载体中,荧光显微镜证实重组体转染PBMCs细胞48 h后可有效表达绿色荧光。显微镜观察发现pEGFP-C3-TCR BV12-3载体转染的PBMCs对肝癌细胞有杀伤作用,但对宫颈癌杀伤作用不明显。结论:成功构建pEGFP-C3-TCR BV12-3表达载体,初步证实TCRBV12-3对肝癌细胞有杀伤作用。更多还原

【Abstract】 Objective: To construct pEGFP-C3-TCR BV12-3 vector, and to investigate the effect of TCR BV12-3 on anti-tumor. Methods: TCR BV12-3 gene was digested from pGEM-T-TCR BV12-3, and was cloned into pEGFP-C3 vector. The pEGFP-C3-TCR BV12-3 recombinant vector was transfected into peripheral blood mononuclear cells(PBMCs) via liposome. After 48 h, the transfection efficiency was measured by fluorescence microscope. Then, the PBMCs, pEGFP-C3-TCR BV12-3 and pEGFP-C3 transfection were co-cultured with hepatocarcinoma cells BEL-7402 and cervical cancer cells HeLa, respectively. After 24 h incubation, the anti-tumor effect of TCR BV12-3 was investigated by microscope. Results: The DNA sequencing analysis showed that TCR BV12-3 gene was successfully subcloned into pEGFP-C3 vector, and the green fluorescence could be detected by fluorescence microscope in PBMCs transfected with pEGFP-C3-TCR BV12-3. PBMCs transfected with pEGFP-C3-TCR BV12-3 showed cytotoxicity in BEL-7402 cells. Conclusion: The pEGFP-C3-TCR BV 12-3 vector was successfully constructed, and the anti-tumor effect of TCR BV12-3 was proved initially.更多还原