摘要
Objective:The aim of this study was to study the effect of human papilloma virus(HPV) type 16 E7 protein expression on growth of RMA cells in vitro and in vivo.Methods:The recombination vector pcDNA3.1-E7 carrying wild type HPV 16 E7 was identified by sequencing.The recombination vector pcDNA3.1-E7 was transfected into mouse lymphadenoma cell line RMA by liposome,and the monoclonal cells transfected stably were obtained by antibiotics G418 sieving and limiting dilution assay.RT-PCR method was used to detect the expression of HPV 16 E7 mRNA in RMA-E7 cells.The growth of RMA cells and RMA-E7 cells cultured in vitro was tested by Cell Count Kit-8.RMA-E7 cells and RMA cells were subcutaneously inoculated in syngeneic mice respectively,the tumor size was measured by sliding caliper twice a week,and the E7 protein expression in tumor tissue of mice was detected by Western blot after tumor formation.The kinetics of cytolytic activity of E7 specific T cells in tumor-bearing mice was measured by LDH kit.Results:Sequencing of recombination vector showed the target gene which was inserted into the recombinant was correct,and RMA-E7 cells expressing E7 protein stably were obtained by limited dilution assay.There were no obvious differences in morphous and growth velocity between RMA cells and RMA-E7 cells in vitro.RMA-E7 cells grew in syngeneic mice were significantly slower than RMA cells.The E7 protein was expressed stronger in RMA-E7 cells in vivo than in vitro.The cytolytic ability of E7-specific CTL was activated at the early stage,reached the maximum at the middle stage,and lost at the end stage.RMA-E7 cells isolated from the tumor-bearing mice were more resistant to E7-specific CTL killing than RMA-E7 cells cultured in vitro.Conclusion:The E7 protein expression has no obvious influence on growth of RMA-E7 cells in vitro,and can suppress growth of RMA-E7 cells in vivo.The activity curve of E7 specific CTL approximately presents "bell" shape.The RMA-E7 cells grew in vivo had a high expression levels of E7 protein,and more resistant to E7-specific CTL killing than those cultured in vitro.The E7 protein expression in vivo not only initiates immune activation,but also induces immune tolerance.
Objective： The aim of this study was to study the effect of human papilloma virus （HPV） type 16 E7 protein ex- pression on growth of RMA cells in vitro and in vivo. Methods： The recombination vector pcDNA3.1-E7 carrying wild type HPV 16 E7 was identified by sequencing. The recombination vector pcDNA3.1-E7 was transfected into mouse lymphadenoma cell line RMA by liposome, and the monoclonal cells transfected stably were obtained by antibiotics G418 sieving and limiting dilution assay. RT-PCR method was used to detect the expression of HPV 16 E7 mRNA in RMA-E7 cells. The growth of RMA cells and RMA-E7 cells cultured in vitro was tested by Cell Count Kit-8. RMA-E7 cells and RMA cells were subcutaneously inoculated in syngeneic mice respectively, the tumor size was measured by sliding caliper twice a week, and the E7 protein expression in tumor tissue of mice was detected by Western blot after tumor formation. The kinetics of cytolytic activity of E7 specific T cells in tumor-bearing mice was measured by LDH kit. Results： Sequencing of recombination vector showed the target gene which was inserted into the recombinant was correct, and RMA-E7 cells expressing E7 protein stably were obtained by limited dilution assay. There were no obvious differences in morphous and growth velocity between RMA cells and RMA-E7 cells in vitro. RMA-E7 cells grew in syngeneic mice were significantly slower than RMA cells. The E7 protein was ex- pressed stronger in RMA-E7 cells in vivo than in vitro. The cytolytic ability of ET-specific CTL was activated at the early stage, reached the maximum at the middle stage, and lost at the end stage. RMA-E7 cells isolated from the tumor-bearing mice were more resistant to E7-specific CTL killing than RMA-E7 cells cultured in vitro. Conclusion： The E7 protein expression has no obvious influence on growth of RMA-E7 cells in vitro, and can suppress growth of RMA-E7 cells in vivo. The activity curve of E7 specific CTL approximately presents ＂bell＂ shape. The RMA-E7 cells grew in vivo had a high expression levels of E7 protein, and more resistant to E7-specific CTL killing than those cultured in vitro. The E7 protein expression in vivo not only initiates immune activation, but also induces immune tolerance.
