The objectives of this study were to investigate the effects of the CRISPR/Cas9 system mediated by the HPV pseudotype virus on SiHa cytobiology behavior by cutting the HPV16 E6 gene selectively and to explore the role...The objectives of this study were to investigate the effects of the CRISPR/Cas9 system mediated by the HPV pseudotype virus on SiHa cytobiology behavior by cutting the HPV16 E6 gene selectively and to explore the role of this system in the treatment of cervical cancer. After designing specific gRNA sequences targeting HPV 16 E6, generating hCas9-EGFP and E6-gRNA-RFP plasmids, and preparing the pseudovirus of HPV16 carrying E6-gRNA and Cas9 plasmids, we determined the titer of the pseudotype virus using the TCID50 method. We obtained the pseudotype virus of HPV16 carrying E6-gRNA and Cas9 plasmids to transfect cervical cancer SiHa cells. Experimental subjects were divided into control group, empty virus group, E6-gRNA transfected group, Cas9 transfected group and Cas9＋E6-gRNA transfected group. The molecular size of the cutting sequence was detected using the T7E1 enzyme digestion method and agarose gel electrophoresis, and the cleavage function of CRISPR/Cas9 on the E6 gene was determined at the same time. RT-PCR and Western blotting were performed to detect the mRNA and protein expression levels of E6 in all the groups; the Transwell cell migration assay was performed to detect the cell migration ability and metastasis in all groups. Heterotopic transplantation tumors were incorporated into mice and were used to investigate the effects of the CRISPR/Cas9 system mediated by the HPV pseudovirus on the tumorigenic ability of SiHa cells by selectively cutting HPV16 E6. The HPV16 pseudotype virus carrying E6-gRNA and Cas9 plasmids could successfully infect SiHa cells, and there were two cutting zones in the Cas9＋E6- gRNA transfected group. However, the empty virus group, E6-gRNA transfected group and Cas9 transfected group had no corresponding zone. Compared with those in the control group, the empty virus group, E6-gRNA transfected group and Cas9 transfected group, the mRNA and protein expression levels of E6 in SiHa cells were downregulated in the Cas9＋E6-gRNA transfected group （P〈0.01）. In addition, the proliferation and migration abilities of SiHa cells were significantly inhibited （P〈0.01）. There were no significant differences among the other groups. In contrast to the control group, the HPV pseudotype virus carrying E6-gRNA and Cas9 plasmids could significantly delay the growth of tumor cells of the ectopic tumor transplantation model （P〈0.01）. The CRISPR/Cas9 system mediated by the HPV pseudotype virus to knockout E6 gene expression exhibited a clear inhibitory effect on the biological function of SiHa cells, which indicated that knocking out the E6 gene using the CRISPR/Cas9 system mediated by the HPV pseudotype virus had a potential effect of eliminating HPV infection and inhibiting the growth of HPV-related tumors. Taken together, these findings provide insight into a new treatment strategy for the prevention and treatment of hr-HPV infected disease, particularly in HPV-related tumors.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
The infections of human papilloma virus (HPV),herpes simplex virus type 2 (HSV-2) and Epstein-Barr virus (EBV) to human cervical epithelium are universal and some of them are associated with the development of cervica...The infections of human papilloma virus (HPV),herpes simplex virus type 2 (HSV-2) and Epstein-Barr virus (EBV) to human cervical epithelium are universal and some of them are associated with the development of cervical carcinomas. One hundred and eleven cervical cancer biopsies taken from Beijing area and Xiangyuan county in Shanxi province of China were examined for the presence of DNA sequences of HPV, HSV-2 and EBV by means of Dot blot hybridization. The experiment results showed that the total infectious rates of HPV, HSV-2 and EBV were 71.17% (79/111), 14.44%(16/111) and 15.63% (15/96), respectively. Seventy-nine samples positive for HPV were further analysed for the viral types distribution, the result indicated that the positive specimens of HPV type 16 accounted for 72.15%, otherwise, the biopsies positive for HPV type 18and 6B/11 only accounted for 16-46% and 11.39%,respectively. The data suggested that HPV infection,especially HPV type 16, may play an important role in the development of cervical carcinomas- 16 specimens positive for HSV-2 were examined for HPV DNAsequences and the result uncovered that 13 out of them were HPV16 positive (81.25%), 11 samples containing EBV genomes were also examined for HPV DNA sequences and the result indicated that 9 of 11 were detectable for HPV DNA. The experiment results proved a direct evtidence of multi-virus infection in cervix and of the synergistic interaction among viruses in the process of cervical epithelial carcinogenesis.Comparing of the viruses' infection of two areas, the frequencies of HPV infection in Beijing and Xiangyuan areas were 72.84% (59/ 81) and 66.67% (20/30) , the infectious rates of HSV-2 in the two areas were 8.64%(7/81) and 30% (9/30) (P<0.05), the rates of EBV infection in the two areas were 12.5% (10/80) and 31.25% (5/16)(0.1>P>0.05). The results proved another strong evidence that the high incidence of cervical cancer in Xiangyuan county may be closely correlated with multiviruses infection and with multi-virus synergistic interaction.展开更多
Epstein-Barr virus was considered as a caustive agent for Burrkitt' s lymphoma and non-malignant B lymphocytes proliferation. The recent studies revealed the striking association of the Infection of EBV with the d...Epstein-Barr virus was considered as a caustive agent for Burrkitt' s lymphoma and non-malignant B lymphocytes proliferation. The recent studies revealed the striking association of the Infection of EBV with the development of human epithelial tumors. 43 specimens of normal exfoliated cervical epithelial cells, 47 biopsies of chronic cervlcitis and 80 tissue samples of cervical carcinomas were tested for the presences of EBV W fragments by using dot blot hybridization method. The results showed that the detectable rates of EBV DNA sequences In the normal exfoliated epithelium, the chronic cervlcitis and cervical carcinomas were 44.16%, 12.77% and 13.75%, respectively. Eleven EBV positive DNA samples from cervical cancers were also examined for the presence of HPV DNA. The result showed 9 out of 11 were HPV DNA positive, the cultanious infectious rate of both viruses was about 81.81%.In this paper, the EBV genomes existed In the part of biopsies of cervical carcinomas were first reported. The results Implied that the development of cervical cancer might be associated with asymptomatic EBV Infection In early stage and might be the result from the synergistic carcinogenesis of EBV with other cervical oncovinises, especially with HPV.展开更多
Introduction: The incidence of head and neck cancers has increased markedly over the last decade. A Danish study of the costs of head and neck cancers has not been undertaken. Such studies have again become relevant d...Introduction: The incidence of head and neck cancers has increased markedly over the last decade. A Danish study of the costs of head and neck cancers has not been undertaken. Such studies have again become relevant due to the development of the HPV vaccines, as some cases are attributable to high-risk HPV 16 or 18. The objective of study was to estimate the incidence of head and neck cancers and their health care costs. Methods: Data on incidence and health care use related to head and neck cancer were obtained from Danish health care registers. New cancer patients were identified in the Danish National Cancer Register. Resource use per year in the hospital sector was estimated using data from the National Patient Register applying charges as cost estimates. Health care consumption by cancer patients was compared with that by an age- and sex-matched cohort without cancer. Results: We found that nearly 1000 new cases of oral cavity, oropharyngeal, hypopharyngeal and laryngeal cancer are diagnosed annually. In total the cost of these cancers to the Danish hospital sector constituted 31.6 million Euros per year, with the majority of costs (74%) occurring in men. The total costs associated with HPV16/18-related head and neck cancers were estimated to be 6.1 million Euros per year. Conclusion: This study provides the first Danish estimates of the costs associated with non-cervical and non-genital HPV-related cancers based on very reliable, individual-based data. It is expected that the current HPV vaccination programme will reduce this burden.展开更多
This paper deals with a control strategy through impulsive drug treatment to study the effect of Chemotherapy and CTL activator drug to eradicate the cervical cancer.CTL kills the HPV infected cells and helps to preve...This paper deals with a control strategy through impulsive drug treatment to study the effect of Chemotherapy and CTL activator drug to eradicate the cervical cancer.CTL kills the HPV infected cells and helps to prevent cervical cancer.Dendritic cell(DC)plays a crucial role in this disease dynamics in enhancing the act ivity of CTL.To explore these effects,we have formulated a mathematical model consisting of six compartments to describe the interactions between HPV and three classes of cervical cells(susceptible,infected,cancerous),with DC and CTL.Calculating the basic reproduction ratio,we find that there exists a disease-free equilibrium point and an endemic equilibrium point.In our study,we have sltown that though DC enhances the activity of CTL.it te not sufficient to eradicate the disease totally.How we could restrict the cancer development by using double drug in an impulsive way with particular drug doses is being reflected in the results.To illustrate our results,numerical simulations are also presented.展开更多
基金This project was supported by the National Natural Science Foundation of China （No. 81302273）, Health and family Planning Commission of Hubei Province, China （No. WJ2015MB084）, Health Department of Hubei Province, China （ No. 2012Z-Y02）, and Science and Technology Department Support Project of Hubei Province, China （No. 2015BCA313）.
文摘The objectives of this study were to investigate the effects of the CRISPR/Cas9 system mediated by the HPV pseudotype virus on SiHa cytobiology behavior by cutting the HPV16 E6 gene selectively and to explore the role of this system in the treatment of cervical cancer. After designing specific gRNA sequences targeting HPV 16 E6, generating hCas9-EGFP and E6-gRNA-RFP plasmids, and preparing the pseudovirus of HPV16 carrying E6-gRNA and Cas9 plasmids, we determined the titer of the pseudotype virus using the TCID50 method. We obtained the pseudotype virus of HPV16 carrying E6-gRNA and Cas9 plasmids to transfect cervical cancer SiHa cells. Experimental subjects were divided into control group, empty virus group, E6-gRNA transfected group, Cas9 transfected group and Cas9＋E6-gRNA transfected group. The molecular size of the cutting sequence was detected using the T7E1 enzyme digestion method and agarose gel electrophoresis, and the cleavage function of CRISPR/Cas9 on the E6 gene was determined at the same time. RT-PCR and Western blotting were performed to detect the mRNA and protein expression levels of E6 in all the groups; the Transwell cell migration assay was performed to detect the cell migration ability and metastasis in all groups. Heterotopic transplantation tumors were incorporated into mice and were used to investigate the effects of the CRISPR/Cas9 system mediated by the HPV pseudovirus on the tumorigenic ability of SiHa cells by selectively cutting HPV16 E6. The HPV16 pseudotype virus carrying E6-gRNA and Cas9 plasmids could successfully infect SiHa cells, and there were two cutting zones in the Cas9＋E6- gRNA transfected group. However, the empty virus group, E6-gRNA transfected group and Cas9 transfected group had no corresponding zone. Compared with those in the control group, the empty virus group, E6-gRNA transfected group and Cas9 transfected group, the mRNA and protein expression levels of E6 in SiHa cells were downregulated in the Cas9＋E6-gRNA transfected group （P〈0.01）. In addition, the proliferation and migration abilities of SiHa cells were significantly inhibited （P〈0.01）. There were no significant differences among the other groups. In contrast to the control group, the HPV pseudotype virus carrying E6-gRNA and Cas9 plasmids could significantly delay the growth of tumor cells of the ectopic tumor transplantation model （P〈0.01）. The CRISPR/Cas9 system mediated by the HPV pseudotype virus to knockout E6 gene expression exhibited a clear inhibitory effect on the biological function of SiHa cells, which indicated that knocking out the E6 gene using the CRISPR/Cas9 system mediated by the HPV pseudotype virus had a potential effect of eliminating HPV infection and inhibiting the growth of HPV-related tumors. Taken together, these findings provide insight into a new treatment strategy for the prevention and treatment of hr-HPV infected disease, particularly in HPV-related tumors.
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.
文摘The infections of human papilloma virus (HPV),herpes simplex virus type 2 (HSV-2) and Epstein-Barr virus (EBV) to human cervical epithelium are universal and some of them are associated with the development of cervical carcinomas. One hundred and eleven cervical cancer biopsies taken from Beijing area and Xiangyuan county in Shanxi province of China were examined for the presence of DNA sequences of HPV, HSV-2 and EBV by means of Dot blot hybridization. The experiment results showed that the total infectious rates of HPV, HSV-2 and EBV were 71.17% (79/111), 14.44%(16/111) and 15.63% (15/96), respectively. Seventy-nine samples positive for HPV were further analysed for the viral types distribution, the result indicated that the positive specimens of HPV type 16 accounted for 72.15%, otherwise, the biopsies positive for HPV type 18and 6B/11 only accounted for 16-46% and 11.39%,respectively. The data suggested that HPV infection,especially HPV type 16, may play an important role in the development of cervical carcinomas- 16 specimens positive for HSV-2 were examined for HPV DNAsequences and the result uncovered that 13 out of them were HPV16 positive (81.25%), 11 samples containing EBV genomes were also examined for HPV DNA sequences and the result indicated that 9 of 11 were detectable for HPV DNA. The experiment results proved a direct evtidence of multi-virus infection in cervix and of the synergistic interaction among viruses in the process of cervical epithelial carcinogenesis.Comparing of the viruses' infection of two areas, the frequencies of HPV infection in Beijing and Xiangyuan areas were 72.84% (59/ 81) and 66.67% (20/30) , the infectious rates of HSV-2 in the two areas were 8.64%(7/81) and 30% (9/30) (P<0.05), the rates of EBV infection in the two areas were 12.5% (10/80) and 31.25% (5/16)(0.1>P>0.05). The results proved another strong evidence that the high incidence of cervical cancer in Xiangyuan county may be closely correlated with multiviruses infection and with multi-virus synergistic interaction.
文摘Epstein-Barr virus was considered as a caustive agent for Burrkitt' s lymphoma and non-malignant B lymphocytes proliferation. The recent studies revealed the striking association of the Infection of EBV with the development of human epithelial tumors. 43 specimens of normal exfoliated cervical epithelial cells, 47 biopsies of chronic cervlcitis and 80 tissue samples of cervical carcinomas were tested for the presences of EBV W fragments by using dot blot hybridization method. The results showed that the detectable rates of EBV DNA sequences In the normal exfoliated epithelium, the chronic cervlcitis and cervical carcinomas were 44.16%, 12.77% and 13.75%, respectively. Eleven EBV positive DNA samples from cervical cancers were also examined for the presence of HPV DNA. The result showed 9 out of 11 were HPV DNA positive, the cultanious infectious rate of both viruses was about 81.81%.In this paper, the EBV genomes existed In the part of biopsies of cervical carcinomas were first reported. The results Implied that the development of cervical cancer might be associated with asymptomatic EBV Infection In early stage and might be the result from the synergistic carcinogenesis of EBV with other cervical oncovinises, especially with HPV.
基金supported by an unrestricted research grant to CAST,University of Southern Denmark,from Sanofi Pasteur MSD.
文摘Introduction: The incidence of head and neck cancers has increased markedly over the last decade. A Danish study of the costs of head and neck cancers has not been undertaken. Such studies have again become relevant due to the development of the HPV vaccines, as some cases are attributable to high-risk HPV 16 or 18. The objective of study was to estimate the incidence of head and neck cancers and their health care costs. Methods: Data on incidence and health care use related to head and neck cancer were obtained from Danish health care registers. New cancer patients were identified in the Danish National Cancer Register. Resource use per year in the hospital sector was estimated using data from the National Patient Register applying charges as cost estimates. Health care consumption by cancer patients was compared with that by an age- and sex-matched cohort without cancer. Results: We found that nearly 1000 new cases of oral cavity, oropharyngeal, hypopharyngeal and laryngeal cancer are diagnosed annually. In total the cost of these cancers to the Danish hospital sector constituted 31.6 million Euros per year, with the majority of costs (74%) occurring in men. The total costs associated with HPV16/18-related head and neck cancers were estimated to be 6.1 million Euros per year. Conclusion: This study provides the first Danish estimates of the costs associated with non-cervical and non-genital HPV-related cancers based on very reliable, individual-based data. It is expected that the current HPV vaccination programme will reduce this burden.
文摘This paper deals with a control strategy through impulsive drug treatment to study the effect of Chemotherapy and CTL activator drug to eradicate the cervical cancer.CTL kills the HPV infected cells and helps to prevent cervical cancer.Dendritic cell(DC)plays a crucial role in this disease dynamics in enhancing the act ivity of CTL.To explore these effects,we have formulated a mathematical model consisting of six compartments to describe the interactions between HPV and three classes of cervical cells(susceptible,infected,cancerous),with DC and CTL.Calculating the basic reproduction ratio,we find that there exists a disease-free equilibrium point and an endemic equilibrium point.In our study,we have sltown that though DC enhances the activity of CTL.it te not sufficient to eradicate the disease totally.How we could restrict the cancer development by using double drug in an impulsive way with particular drug doses is being reflected in the results.To illustrate our results,numerical simulations are also presented.
