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
Using polymerase chain reaction (PCR), 25 specimens from 22 patients with oral carcinomas were examined by 6 selected primers of human papillomavirus (HPV). Eighteen of the 22 patients (18/22) gave positive reaction w...Using polymerase chain reaction (PCR), 25 specimens from 22 patients with oral carcinomas were examined by 6 selected primers of human papillomavirus (HPV). Eighteen of the 22 patients (18/22) gave positive reaction with a positive rate of 81.8%. The positive rates of HPV 6, 11, 16 and 18 were 27.3%, 18.2%, 63.6% and 40.9% respectively. 13.6% positive for mixed infection of HPV 16 and 18 (3/22) and 18.2% positive for mixed infection of HPV, 6, 11, 16 and 18 (4/22). Examining enlarged cervical lymph nodes in three cases with suspecting metastases to cervical lymph nodes from oral carcinomas. It revealed HPV DNA 16 and 18 in two cases and HPV DNA 18 in one case. These results suggested that there was a tendency for HPV 16 and 18 to metastasinze via lymphatics. Only one case of the three had a pathologic diagnosis of lymph node metastasis. Of the 30 non tumor controls, HPV DNA positivity was 10%, all being HPV 18. χ 2 test gave a P<0.005. It strongly indicated that HPV 16 and 18 were related to oral carcinomas.展开更多
Objective:To identify circulating HPV types among urban Indonesian women and their specific co-infection patterns in bid to curb HPV infection in the general population and minimize its complications.Methods:Urban Ind...Objective:To identify circulating HPV types among urban Indonesian women and their specific co-infection patterns in bid to curb HPV infection in the general population and minimize its complications.Methods:Urban Indonesian women from general population were selected as sample framework.Sample size and distribution across regions were determined by the Indonesian Bureau of Statistics(Badan Pusat Statistik,BPS),which represented the national level.Up to 35408 cervical swab specimens were collected from August to September 2016 in 34 Indonesian provinces,categorized into six regions based on the development criteria set by the Ministry of National Development Planning(Badan Perencanaan Pembangunan Nasional,BAPPENAS).From all 1874 samples identified as HPV-positive,hybrid capture was implemented to evaluate type-specific HPV.This study analyzed descriptive data to determine the core-cluster of HPV combination.Co-occurrence HPV network was assessed using‘qgraph’package version 1.6.3 and computed in R version 3.6.3.Two-HPV association was analyzed in logistic regression using bias-reduction generalized linear model(brglm2)package version 0.5.1 adjusted by age and six main Indonesian regions.Results:The logistic regression analysis demonstrated that HPV type 52 had rare relationship despite its common co-occurrence,cementing its role in single HPV infection.HPV type 16 and 18 tended to form infection cluster and were strongly associated with other types.Conclusions:HPV type 52 was the most frequent HPV type among urban Indonesian women and accounted for most single infection cases.Concurrently,HPV 16 and HPV 18 accounted for most multiple infection cases and had strong tendency to attract other types,which may add further complications.However,due to lack of cytology and histological examination and information for other potential determinants,further in-depth studies are necessary to confirm whether these infection patterns truly connect to certain clinical outcomes.展开更多
目的研究慢病毒携带的短发夹状干扰RNA(short hairp in RNA,shRNA)对宫颈癌Cask i细胞中人乳头瘤病毒(hum an pap illom a virus,HPV)16型E6表达的抑制作用。方法将靶向HPV16型E6的shRNA表达序列克隆到改建的慢病毒表达载体PLL3.7中,经...目的研究慢病毒携带的短发夹状干扰RNA(short hairp in RNA,shRNA)对宫颈癌Cask i细胞中人乳头瘤病毒(hum an pap illom a virus,HPV)16型E6表达的抑制作用。方法将靶向HPV16型E6的shRNA表达序列克隆到改建的慢病毒表达载体PLL3.7中,经限制性酶切鉴定和测序后,与3种包装质粒混合,以L ipofectam ine 2000包裹后转染293FT细胞,72 h后收取含病毒的上清液并感染宫颈癌Cask i细胞,感染48 h后加入G418筛选阳性克隆。每天对阳性细胞克隆进行细胞计数;提取细胞总mRNA,进行RT-PCR反应。结果与对照组相比HPV16型E6的表达降低70%,宫颈癌Cask i细胞生长速度减慢50%。结论慢病毒携带的短发夹状干扰RNA能干扰宫颈癌细胞中HPV16型E6的表达并有抑制癌细胞生长的作用。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 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.
文摘Using polymerase chain reaction (PCR), 25 specimens from 22 patients with oral carcinomas were examined by 6 selected primers of human papillomavirus (HPV). Eighteen of the 22 patients (18/22) gave positive reaction with a positive rate of 81.8%. The positive rates of HPV 6, 11, 16 and 18 were 27.3%, 18.2%, 63.6% and 40.9% respectively. 13.6% positive for mixed infection of HPV 16 and 18 (3/22) and 18.2% positive for mixed infection of HPV, 6, 11, 16 and 18 (4/22). Examining enlarged cervical lymph nodes in three cases with suspecting metastases to cervical lymph nodes from oral carcinomas. It revealed HPV DNA 16 and 18 in two cases and HPV DNA 18 in one case. These results suggested that there was a tendency for HPV 16 and 18 to metastasinze via lymphatics. Only one case of the three had a pathologic diagnosis of lymph node metastasis. Of the 30 non tumor controls, HPV DNA positivity was 10%, all being HPV 18. χ 2 test gave a P<0.005. It strongly indicated that HPV 16 and 18 were related to oral carcinomas.
基金Ministry of Health,Republic of Indonesia provided financial assistance to support this study from their annual budget allocation.
文摘Objective:To identify circulating HPV types among urban Indonesian women and their specific co-infection patterns in bid to curb HPV infection in the general population and minimize its complications.Methods:Urban Indonesian women from general population were selected as sample framework.Sample size and distribution across regions were determined by the Indonesian Bureau of Statistics(Badan Pusat Statistik,BPS),which represented the national level.Up to 35408 cervical swab specimens were collected from August to September 2016 in 34 Indonesian provinces,categorized into six regions based on the development criteria set by the Ministry of National Development Planning(Badan Perencanaan Pembangunan Nasional,BAPPENAS).From all 1874 samples identified as HPV-positive,hybrid capture was implemented to evaluate type-specific HPV.This study analyzed descriptive data to determine the core-cluster of HPV combination.Co-occurrence HPV network was assessed using‘qgraph’package version 1.6.3 and computed in R version 3.6.3.Two-HPV association was analyzed in logistic regression using bias-reduction generalized linear model(brglm2)package version 0.5.1 adjusted by age and six main Indonesian regions.Results:The logistic regression analysis demonstrated that HPV type 52 had rare relationship despite its common co-occurrence,cementing its role in single HPV infection.HPV type 16 and 18 tended to form infection cluster and were strongly associated with other types.Conclusions:HPV type 52 was the most frequent HPV type among urban Indonesian women and accounted for most single infection cases.Concurrently,HPV 16 and HPV 18 accounted for most multiple infection cases and had strong tendency to attract other types,which may add further complications.However,due to lack of cytology and histological examination and information for other potential determinants,further in-depth studies are necessary to confirm whether these infection patterns truly connect to certain clinical outcomes.
文摘目的研究慢病毒携带的短发夹状干扰RNA(short hairp in RNA,shRNA)对宫颈癌Cask i细胞中人乳头瘤病毒(hum an pap illom a virus,HPV)16型E6表达的抑制作用。方法将靶向HPV16型E6的shRNA表达序列克隆到改建的慢病毒表达载体PLL3.7中,经限制性酶切鉴定和测序后,与3种包装质粒混合,以L ipofectam ine 2000包裹后转染293FT细胞,72 h后收取含病毒的上清液并感染宫颈癌Cask i细胞,感染48 h后加入G418筛选阳性克隆。每天对阳性细胞克隆进行细胞计数;提取细胞总mRNA,进行RT-PCR反应。结果与对照组相比HPV16型E6的表达降低70%,宫颈癌Cask i细胞生长速度减慢50%。结论慢病毒携带的短发夹状干扰RNA能干扰宫颈癌细胞中HPV16型E6的表达并有抑制癌细胞生长的作用。
