In order to provide the experimental basis for the 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 ...In order to provide the experimental basis for the 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 3MP-1-macrophages, and the reconstructed plasmid pcDNA3.1 （-）/E6 was transfected into THP-1-macrophages. The expression of E6 gene was assayed in macrophage lysates by using Western blot analysis and the level of TNF-α or IL-1β was examined by ELISA. All of data were analyzed by SPSS12.0. As demonstrated by Western blot analysis, the expression of E6 protein with a molecular weight of about 18 kDa by plasmid pcDNA3.1 （-）/E6 in THP-1-macrophages could be detected. However, as demonstrated by ELISA assay, the level of TNF-α or IL-1β 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）. All these results illustrate that the transient over-expression of HPV6 E6 protein reduces the production of TNF-α and IL-1β induced by LPS in THP-1-macrophages.展开更多
Objective: To explore the clinical significance of the quantitative detection of human papillomavirus(HPV) E6/E7 mRN A in triage of patients with atypical squamous cells of undetermined significance(ASC-US) and low-gr...Objective: To explore the clinical significance of the quantitative detection of human papillomavirus(HPV) E6/E7 mRN A in triage of patients with atypical squamous cells of undetermined significance(ASC-US) and low-grade squamous intraepithelial lesion(LSIL).Methods: A cross-sectional screening study was conducted among women who underwent outpatient gynecological screening at the Obstetrics and Gynecology Hospital of Fudan University from September 2015 to July 2016. A total of 500 patients from our hospital with ASC-US or LSIL based on cytology testing were subjected to HPV DNA and HPV E6/E7 mRNA quantitative analysis.Results: The specificity of the HPV E6/E7 mRNA test for detecting ≥high-grade squamous intraepithelial lesion(HSIL+) was statistically higher than that of the HPV DNA test(61.3% vs. 40.0%, P< 0.05), whereas there was no significant difference in the sensitivity of HPV E6/E7 mRNA test and HPV DNA test(90.0% vs. 95.0%, P > 0.05). The positive rates of HPV in the participants tested by HPV E6/E7 mRNA and HPV DNA were, respectively, 42.8%(214/500) and 62.8%(314/500), with statistical significance(P < 0.05).Conclusions: The HPV E6/E7 mRNA test was slightly less sensitivity than that of the HPV DNA test for diagnosing HSIL+ in patients with ASC-US and LSIL, but the difference was not significant, although the specificity of the former was significantly higher. HPV E6/E7 mRNA detection can effectively reduce overdiagnosis and overtreatment of patients with ASC-US and LSIL and has important clinical value in triage of patients with ASC-US and LSIL.展开更多
Background Heat shock protein 70 （HSP70） is expressed highly in epithelial tumours associated closely with human papillomavirus 16 （HPV16） infections. However, evidence about the direct relationship between HSP70 ...Background Heat shock protein 70 （HSP70） is expressed highly in epithelial tumours associated closely with human papillomavirus 16 （HPV16） infections. However, evidence about the direct relationship between HSP70 expression and HPVs infections are still lacking. In the present study, we examined the expression of HSP70 in keratinocytes introduced with HPV16 E6/E7 oncogenes. Methods Stable transfected cells were established by transfection of the plasmids pLXSN16E6/E7 into cultured primary keratinocytes and subsequently selected by plasmid specific selection antibiotic （G418） at the required concentration. The expression of HSP70 in pLXSN16E6/E7 transfected keratinocytes was determined by Western blot. The correlation of HSP70 expression and E6/E7 transfeetion was further confirmed by doubly labelled immunofluorescent staining. Results Compared to non-transfected keratinocytes, there was a significant trend for higher levels of HSP70 in pLXSN16E6/E7 transfected keratinocytes. Doubly labelled immunofluorescent staining experiment showed that the co-localization of HPV16 E6/E7 and HSP70 in transfeeted keratinoeytes was observed and increased expression of HSP70 was strongly associated with the transfection of HPV16 E6/E7. Conclusions Our studies demonstrated increased levels of HSP70 proteins in keratinocytes stably transfected by HPV16 E6/E7 oncogenes. It suggests that the expression of HSP70 is modulated by HPV16 E6/E7 proteins, which may be involved in HPV16 E6/E7 induced immortalization.展开更多
文摘In order to provide the experimental basis for the 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 3MP-1-macrophages, and the reconstructed plasmid pcDNA3.1 （-）/E6 was transfected into THP-1-macrophages. The expression of E6 gene was assayed in macrophage lysates by using Western blot analysis and the level of TNF-α or IL-1β was examined by ELISA. All of data were analyzed by SPSS12.0. As demonstrated by Western blot analysis, the expression of E6 protein with a molecular weight of about 18 kDa by plasmid pcDNA3.1 （-）/E6 in THP-1-macrophages could be detected. However, as demonstrated by ELISA assay, the level of TNF-α or IL-1β 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）. All these results illustrate that the transient over-expression of HPV6 E6 protein reduces the production of TNF-α and IL-1β induced by LPS in THP-1-macrophages.
基金supported by the Shanghai Science and Technology Committee Project(No.16411950200).
文摘Objective: To explore the clinical significance of the quantitative detection of human papillomavirus(HPV) E6/E7 mRN A in triage of patients with atypical squamous cells of undetermined significance(ASC-US) and low-grade squamous intraepithelial lesion(LSIL).Methods: A cross-sectional screening study was conducted among women who underwent outpatient gynecological screening at the Obstetrics and Gynecology Hospital of Fudan University from September 2015 to July 2016. A total of 500 patients from our hospital with ASC-US or LSIL based on cytology testing were subjected to HPV DNA and HPV E6/E7 mRNA quantitative analysis.Results: The specificity of the HPV E6/E7 mRNA test for detecting ≥high-grade squamous intraepithelial lesion(HSIL+) was statistically higher than that of the HPV DNA test(61.3% vs. 40.0%, P< 0.05), whereas there was no significant difference in the sensitivity of HPV E6/E7 mRNA test and HPV DNA test(90.0% vs. 95.0%, P > 0.05). The positive rates of HPV in the participants tested by HPV E6/E7 mRNA and HPV DNA were, respectively, 42.8%(214/500) and 62.8%(314/500), with statistical significance(P < 0.05).Conclusions: The HPV E6/E7 mRNA test was slightly less sensitivity than that of the HPV DNA test for diagnosing HSIL+ in patients with ASC-US and LSIL, but the difference was not significant, although the specificity of the former was significantly higher. HPV E6/E7 mRNA detection can effectively reduce overdiagnosis and overtreatment of patients with ASC-US and LSIL and has important clinical value in triage of patients with ASC-US and LSIL.
基金This work was supported by a grant from the National NaturalScience Foundation of China (No.39570656)
文摘Background Heat shock protein 70 （HSP70） is expressed highly in epithelial tumours associated closely with human papillomavirus 16 （HPV16） infections. However, evidence about the direct relationship between HSP70 expression and HPVs infections are still lacking. In the present study, we examined the expression of HSP70 in keratinocytes introduced with HPV16 E6/E7 oncogenes. Methods Stable transfected cells were established by transfection of the plasmids pLXSN16E6/E7 into cultured primary keratinocytes and subsequently selected by plasmid specific selection antibiotic （G418） at the required concentration. The expression of HSP70 in pLXSN16E6/E7 transfected keratinocytes was determined by Western blot. The correlation of HSP70 expression and E6/E7 transfeetion was further confirmed by doubly labelled immunofluorescent staining. Results Compared to non-transfected keratinocytes, there was a significant trend for higher levels of HSP70 in pLXSN16E6/E7 transfected keratinocytes. Doubly labelled immunofluorescent staining experiment showed that the co-localization of HPV16 E6/E7 and HSP70 in transfeeted keratinoeytes was observed and increased expression of HSP70 was strongly associated with the transfection of HPV16 E6/E7. Conclusions Our studies demonstrated increased levels of HSP70 proteins in keratinocytes stably transfected by HPV16 E6/E7 oncogenes. It suggests that the expression of HSP70 is modulated by HPV16 E6/E7 proteins, which may be involved in HPV16 E6/E7 induced immortalization.
