Objective To evaluate the overexpression of cyclin G1 in cervical intraepithelial neoplasia （CIN） and cervical carcinoma, and the correlation between cyclin G1 and high-risk human papilloma virus （HPV） infection. ...Objective To evaluate the overexpression of cyclin G1 in cervical intraepithelial neoplasia （CIN） and cervical carcinoma, and the correlation between cyclin G1 and high-risk human papilloma virus （HPV） infection. Methods All of the specimens were obtained from the Department of Pathology of China-Japan Friendship Hospital from January 2000 to August 2004. We detected the expression of cyclin G1 with immunohistochemistry, HPV16/18 infection with in situ hybridization, and high-risk HPV infection with Hybrid capture system Ⅱ （HC-Ⅱ） in normal group （25 cases）, CIN Ⅰ （48 cases）, CIN Ⅱ （56 cases）, CIN Ⅲ（54 cases）, and invasive cervical squamous-cell carcinoma （SCC, 31 cases）. Results The positive rates of cyclin G1 expression in CIN（77.85% ）and SCC cervical tissues （87. 10% ） were significantly higher than normal （ 8.00%, P 〈 0. 01 ）, and the intensities of cyclin G1 expression in CIN（40. 60% ） and SCC cervical tissues （61.51%） were significantly higher than normal （2. 72%, P 〈0.05）. The positive rates and intensities of cyclin G1 expression increased gradually with the grade of cervical lesions. High-risk HPV infection rates were higher in CIN and SCC than normal groups （P 〈 0.05 ）. There was a positive correlation between cyclin G1 expression and high-risk HPV infection detected with HC-Ⅱ （Kendall＇s tau-b =0. 316, 0. 269, 0. 352, and 0. 474 in CIN Ⅰ, CIN Ⅱ, CIN Ⅲ, and SCC, respectively, P 〈 0. 05 ）. Conclusions Cyclin G1 is overexpressed in CIN and SCC. Cyclin G1 may be a biomarker for detecting CIN and SCC. Cyclin G1 may play an important role in the oncogenesis of CIN and SCC by high-risk HPV infection.展开更多
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 ca...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.展开更多
An optimized recombinant HPV16 E6E7 fusion gene (HPV16 ofE6E7) was constructed according to codon usage for mammalian cell expression, and a mutant of HPV16 ofE6E7 fusion gene (HPV16 omfE6E7) was generated by site-dir...An optimized recombinant HPV16 E6E7 fusion gene (HPV16 ofE6E7) was constructed according to codon usage for mammalian cell expression, and a mutant of HPV16 ofE6E7 fusion gene (HPV16 omfE6E7) was generated by site-directed mutagenesis at L57G, C113R for the E6 protein and C24G, E26G for the E7 protein for HPV16 ofE6E7 [patent pending (CN 101100672)]. The HPV16 omfE6E7 gene constructed in this work not only lost the transformation capability to NIH 3T3 cells and tumorigenicity in SCID mice, but also maintained very good stability and antigenicity. These results suggests that the HPV16 omfE6E7 gene should undergo further study for application as a safe antigen-specific therapeutic vaccine for HPV16-associated tumors.展开更多
文摘Objective To evaluate the overexpression of cyclin G1 in cervical intraepithelial neoplasia （CIN） and cervical carcinoma, and the correlation between cyclin G1 and high-risk human papilloma virus （HPV） infection. Methods All of the specimens were obtained from the Department of Pathology of China-Japan Friendship Hospital from January 2000 to August 2004. We detected the expression of cyclin G1 with immunohistochemistry, HPV16/18 infection with in situ hybridization, and high-risk HPV infection with Hybrid capture system Ⅱ （HC-Ⅱ） in normal group （25 cases）, CIN Ⅰ （48 cases）, CIN Ⅱ （56 cases）, CIN Ⅲ（54 cases）, and invasive cervical squamous-cell carcinoma （SCC, 31 cases）. Results The positive rates of cyclin G1 expression in CIN（77.85% ）and SCC cervical tissues （87. 10% ） were significantly higher than normal （ 8.00%, P 〈 0. 01 ）, and the intensities of cyclin G1 expression in CIN（40. 60% ） and SCC cervical tissues （61.51%） were significantly higher than normal （2. 72%, P 〈0.05）. The positive rates and intensities of cyclin G1 expression increased gradually with the grade of cervical lesions. High-risk HPV infection rates were higher in CIN and SCC than normal groups （P 〈 0.05 ）. There was a positive correlation between cyclin G1 expression and high-risk HPV infection detected with HC-Ⅱ （Kendall＇s tau-b =0. 316, 0. 269, 0. 352, and 0. 474 in CIN Ⅰ, CIN Ⅱ, CIN Ⅲ, and SCC, respectively, P 〈 0. 05 ）. Conclusions Cyclin G1 is overexpressed in CIN and SCC. Cyclin G1 may be a biomarker for detecting CIN and SCC. Cyclin G1 may play an important role in the oncogenesis of CIN and SCC by high-risk HPV infection.
文摘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.
基金National 863 Program(2007AA021107)Beijing Municipal Commission of Education Research Projects(KM201010005008)Doctoral Start-up Foundation of Beijing University of Technology
文摘An optimized recombinant HPV16 E6E7 fusion gene (HPV16 ofE6E7) was constructed according to codon usage for mammalian cell expression, and a mutant of HPV16 ofE6E7 fusion gene (HPV16 omfE6E7) was generated by site-directed mutagenesis at L57G, C113R for the E6 protein and C24G, E26G for the E7 protein for HPV16 ofE6E7 [patent pending (CN 101100672)]. The HPV16 omfE6E7 gene constructed in this work not only lost the transformation capability to NIH 3T3 cells and tumorigenicity in SCID mice, but also maintained very good stability and antigenicity. These results suggests that the HPV16 omfE6E7 gene should undergo further study for application as a safe antigen-specific therapeutic vaccine for HPV16-associated tumors.
