摘要
目的将人乳头瘤病毒16型(HPV 16)E6蛋白真核表达载体pcDNA3.1(-)/E6转染巨噬细胞,观察E6蛋白瞬时高表达对LPS诱导的THP-1巨噬细胞分泌TNF-α和IL-1β的影响,为进一步研究HPV16E6蛋白的致病机制奠定实验基础。方法将质粒pcDNA3.1(-)/E6通过SuperFectTM脂转染试剂转染巨噬细胞24h后,用LPS刺激巨噬细胞,利用Western-blotting鉴定E6蛋白在巨噬细胞中的表达,ELISA检测pcDNA3.1(-)/E6转染的巨噬细胞不同时间(12、24及48h)培养上清液中TNF-α与IL-1β的含量,统计软件SPSS12.0分析所得数据。结果Western-blotting结果显示pcDNA3.1(-)/E6能在巨噬细胞中表达约18kD的目的蛋白,即E6蛋白;pcDNA3.1(-)/E6+LPS组培养上清中TNF-α与IL-1β的量明显低于巨噬细胞+LPS组和pcDNA3.1(-)+LPS组,pcDNA3.1(-)/E6+LPS组与巨噬细胞+LPS组和pcDNA3.1(-)+LPS组比较,差异具有显著性(P<0.01)。结论HPV16E6蛋白瞬时高表达下调LPS诱导的THP-1巨噬细胞分泌TNF-α与IL-1β。
[Objective] In order to provide the experimental basis for further studies on the oncogenic mechanism of the E6 protein from human papillomavirus type 16 （HPV16）, the eukaryotic expression vector pcDNA3.1 （-）/E6 was used for the study on the effect of E6 protein to influence the secretory activity of LPS-induced THP-1 macrophages. [Methods] The reconstructed plasmid pcDNA3.1 （-）/E6 was transfected into THP-1 macrophages. The expression of E6 gene was assayed in macrophages lysates by using Western blot analysis and the level of TNF-α or IL-1β was examined by ELISA, respectively. All of data were analyzed by SPSS 12.0. [Results] The expression of E6 protein with a molecular weight of about 18 kD by plasmid pcDNA3.1 （-）/E6 in THP-1 macrophages could be detected by Western blot analysis. The level of TNF-αand IL-1β induced by LPS in lysates of THP-1 macrophages showed an obvious difference between the pcDNA3.1 （-）/E6 group and the LPS control group or the pcDNA3.1 （-） control group （P 〈0.01）, but no significant difference existed between pcDNA3.1 （-） control group and LPS control group （P 〉0.05） by ELISA. [Conclusion] The E6 protein could express correctly in THP-1 macrophages. The transient over-expression of HPV6 E6 protein reduces the production of TNF-α and IL-1β induced by LPS in THP-1-macrophages.
出处
《中国现代医学杂志》
CAS
CSCD
北大核心
2008年第13期1845-1848,共4页
China Journal of Modern Medicine
基金
湖南省教育厅资助项目(No:02C391)
