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 wil...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.展开更多
文摘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.
