重组杆状病毒载体介导报告基因在甲状腺癌细胞中的表达研究

Experimental study of baculovirus-mediated reporter gene expression in thyroid carcinoma cells

目的探讨杆状病毒作为甲状腺癌基因转移和治疗载体的可行性。方法利用杆状病毒Bac-to-Bac表达系统制备携带绿色荧光蛋白(GFP)的重组杆状病毒(Bac-GFP)。分别以不同感染复数(MOI)的Bac-GFP感染滤泡状甲状腺癌细胞株FTC-133,流式细胞仪鉴定感染效率及GFP表达强度。以MOI为100的Bac-GFP感染FTC-133,荧光显微镜观察GFP表达持续时间;流式细胞仪检测不同浓度丁酸钠干预对感染效率及GFP表达强度的影响。MTT比色法检测不同MOI的Bac-GFP感染对FTC-133细胞存活的影响。结果成功制备重组杆状病毒载体Bac-GFP并感染FTC-133。感染效率随MOI的增加而提高;当MOI为100时,感染效率高达71.3%。丁酸钠可提高Bac-GFP对FTC-133的感染效率及GFP表达强度,但感染效率的提高无统计学意义。荧光显微镜观察显示,Bac-GFP感染FTC-133其GFP表达可持续2周。MTT检测显示,不同MOI的Bac-GFP感染FTC-133的细胞存活率比较差异无统计学意义(P>0 05)。结论重组杆状病毒Bac-GFP对FTC-133细胞具有较高的感染效率,报告基因表达时间较长且无明显细胞毒性,有可能成为甲状腺癌基因治疗的良好载体。

Objective To investigate the feasibility of baculovirus in gene transfer and therapeutic vector for thyroid carcinoma. Methods Recombinant baculovirus carrying green fluorescent protein （GFP） （Bac-GFP） was constructed with baculovirus Bac-to-Bac expression system. Human follicular thyroid carcinoma cell line FTC-133 was infected with Bac-GFP at different multiplicity of infection （ MOI）, and infection efficiency and GFP expression intensity were identified by flow cytometry. FTC-133 cells were infected by Bac-GFP with MOI of 100, and the duration of GFP expression was observed by fluorescence microscopy. The infection efficiency of Bac-GFP and the GFP expression intensity in the presence of different concentrations of sodium butyrate were determined by flow cytometry. FTC-133 cell survival after infection by Bac-GFP with different MOI was detected by MTT assay. Results Baculovirus vector Bac-GFP was successfully constructed, and FTC- 133 cells were infected. The infection efficiency increased with MOI, and reached as high as 71.3% at MOI of 100. Sodium butyrate increased both the infection efficiency and GFP expression intensity, while there was no significant difference in the infection efficiency between the presence and absence of sodium butyrate. Fluorescence microscopy revealed that the expression of GFP in FTC-133 cells infected by Bac-GFP lasted two weeks. MTT assay indicated that there was no significant difference in cell survival among FTC-133 cells infected by Bac-GFP with different MOI （ P 〉 0. 05）. Conclusion Recombinant baculovirus Bac-GFP has a high infection efficiency in FTC-133 cells, and the expression of report gene lasts longer, with no obvious cytotoxicity, which makes it a favourable vector for gene therapy of thyroid carcinoma.