| Condyloma acuminatum (CA) is one of the most common sexually transmitted diseases, which is difficult to cure because of its high relapse rate. The incidence of CA had a tendency of increase. Currently, we usually eliminate the neoplasm by physical therapy, including laser, microwave, liquid nitrogen, etc, followed by non-specific immunotherapy. Routine therapeutic methods can't efficiently eradicate CA in that all existing treatment options have an associated failure rate and recurrence of genital warts after treatment is common.Human papilloma virus (HPV) has been demonstrated to be the most causative pathogen of CA and cervical cancer. It has been identified more than 130 subtypes, among which the HPV6b and HPV11 are responsible for more than 90% of CA.The HPV genome consists of eight kilobasepairs (kbp) and is a double-stranded DNA molecule. A particular characteristic of papillomaviruses is that the partly overlapping ORFs are arranged on only one DNA strand. The genome can be divided into three regions: the long control region (LCR) without coding potential, the region of early proteins (E1-E8) and the region of late protein (L1 and L2). Oncogene E6 and E7 of high-risk types play a crucial role in induction andmaintenance of malignant transformation of HPV-associated lesions, however, both antigens have immune dominant epitopes and therefore represent ideal targets for developments of therapy against HPV.HPVs have the property of strictly epithelial infection, high specificity of host and unable reproduction in routine cell culture, which restrict the research on the biological function and immunological mechanism. Presently researches associated with HPV focus on type 16/18 accounting for 99% cervical cancer, whereas studies on type 6/11 accounting for most of CA are rare.Nowadays biomedical therapy on HPV infection tends to specially inhibiting virus, such as vaccination of DNA- or peptide- vaccine and silencing specific genes by RNA interference. All these strategies depend on the identification of HPV types and mastery of the characteristic of distribution of HPV types in local. The uncertainty of the types of HPV makes it difficult to evaluate HPV infecton and apply type-specific therapeutic approach. It calls for accurate and rapid methods of identification of HPV types.Since natural immune responses to persistent infection of HPV are unable to elicit a sufficient immune protection due to poor immunogenicity of HPV, up-regulation of immune responses is difficult to achieve. Thereafter, how to raise the immunological response to HPV to reduce the relapse rate of CA by host remains a focusing problem. Enhancement of presentation of the HPV antigen to elicit efficiently specific CTL response has been a research emphasis. Calreticulin (CRT), an abundant 46 Kda Ca2+-binding protein which is located in the endoplasmic reticulum (ER), has been recently reported to act as endothelial cell inhibitors of tumor growth and its chaperone effects in cancer vaccines was also identified. CA lesions are characterized by proliferation of micro vessels and vascular hyperemia in dermal papilla, the growth of wart tissue depending on adequate blood supply resulting from new microvessels formation, which is known as angiogenesis.Antiangiogenic factors may suppress adequate blood supply and inhibit CA development. Enhancement of immunogenicity of the target antigen HPV6bE7 and suppression of vessels formation could be two potentially attractive approaches in vaccine strategies to control CA.RNA interference (RNAi) is the process by which double-stranded RNA directs sequence-specific degradation of messenger RNA in animal and plant cells. The process is highly efficient and specific. Selective silencing of gene expression has recently been achieved using siRNA. RNAi is now established as an important biological strategy for gene silencing of target genes. It has been applied in the therapeutic research of viral infection, autoimmune disease, inflammation and tumor. RNAi quickly became a weapon for attack on multiple viruses, such as Hepatitis B virus (HBV) or Hepatitis C virus (HCV), Human immunodeficiency virus (HIV), HPV, Repiratory syncytial virus (RSV) and poliovirus, etc. Here we introduced RNAi into HPV research and asked if siRNA can be employed to silence exogenous viral gene expression of HPV in mammalian cells.Taken together, this project focused on diagnosis, immunological therapy and gene silencing of HPV infection and offered the theoretically basis and references of therapeutic approach of HPV-associated disease.Three sections of this paper were as follows:Section one Polymerase chain reaction mediated by various primers inhuman papillomavirus detectionPolymerase chain reaction (PCR) has been shown to be an approach with high sensitivity and speciality in identifying a certain virus in clinical samples. PCR technique could diagnose and type directly by observation the length of gene fragments. To analyze the prevalence of local, we applied PCR mediated by type-specific primers in detection of the most frequent low-risk types 6, 11 orhigh-risk types 16,18 of HPV in CA samples.It is almost impossible to detect all the HPV types of infection in a single reaction system, our assay of detection of HPV DNA may underestimate the multiple types or rare types of HPV. To determine a feasible and accurate method in detecting HPV infection, we compared and evaluated the efficacy of the two most commonly used PCR general primers: MY09/11 and GP5+/6+ in HPV DNA detection. 1.1 PCR detection mediated by type-specific primers and general primers104 cases of biopsies specimen of CA by clinical and pathological diagnosis and 12 specimens of penis foreskin were obtained from patients in hospital affiliated of Zhejiang University and the tissues were crushed frozen under liquid nitrogen and extracted of genomic DNA by saturated phenol/ chloroform / isopropyl alcohol. The DNA of samples was precipitated with resolved and diluted by ddH2O and amplified by PCR reaction with four pairs of type-specific primers: HPV6/11/16/18 and general primers: MY09/11 and GP5+/6+ respectively. Negative and blank controls were also included. The data showed 101 (97.1%) out of 104 CA specimens were detected positive by type-specific primers, including 51 cases (49.0%) of HPV6, 94 cases (90.4%) of HPV11, 22 cases (21.2%) of HPV16, 13 cases (12.5%) of HPV18. Type-specific primers-mediated PCR showed positive rate of simple HPV 11 infection was markedly higher than that of simple HPV6b infection in local region (P <0.01). It appeared the occurrence of dual-infection of HPV6 as well as HPV 11 had an increasing tendency in recent years, which deserved more attention paid to it. Dual-primers mediated PCR in one reaction system in this assay could identify HPV6 as well as HPV 11 simultaneously.1 cases positive for HPV DNA by general primer but negative for HPV6b, 11, 16, 18 by type-specific primers might be other types beyond HPV6b, 11, 16, 18 and could further be identified if necessary.1.2 The sensitivity of two pairs of general primersThe positive rate of HPV infection of the CA specimens was detected of 98.1% (102 of 104) by using GP5+/6+ primers, which was higher than that of 92.3%(96 of 104) by MY09/11 in CA groups respectively. The positive rate of HPV infection of foreskin groups by two general primers showed a consensus result of 8.3% (1 of 12).The HPV6/11 standard plasmid serially diluted to five different concentrations starting from lOng/^1 was used as template for PCR amplification by MY09/11 and GP5+/6+ primers, respectively. The lowest concentration of positive fragment was sensitivity of general primers. Also, GP5+/6+ mediated-PCR could detect HPV DNA lower than lpg, while MY09/11 mediate-PCR only detected Ing HPV DNA. It showed there was over 3-log difference between two general primers-mediated detection systems in the amplification of HPV DNA. It demonstrated GP5+/6+ could detect even lower concentration than MY09/11 of HPV DNA, indicating the sensitivity of GP5+/6+ was significantly higher than that of MY09/11 in detection HPV DNA of tittle level.Our assay demonstrated the type-specific primers-mediated PCR could offer a feasible method in detecting HPV DNA as well as typing. The general primers GP5+/6+ have higher sensitivity than MY09/11 and accordingly can be applied in screening of positive cases of HPV infection in clinic. Combination with detection by type-specific primers-mediated PCR in positive cases allows the fast and convenient analysis of samples for large-scale epidemiological studies.Section two The immune response and antiangiogenic activity of recombinant DNA vaccines CRT/HPV6bE7With the development of modern molecular biological technology, we constructed HPV DNA vaccines encoding different CRT fragments aa 1-180 or aa 1-120 linked to HPV6bE7 and tried to elucidate their immunological andantiangiogenic effects by using established HPV6bE7-positive B16 melanoma cellline as well as an animal model. The active region of CRT for vaccine developmentwas also determined in the present study. We compared CRT1-120 with CRT1-180fragment trying to find the potent domain with the anti-tumor and anti-angiogeniceffects.2.1 Construction of DNA vaccines and establishment of HPV6bE7-expressingcell lineHPV6bE6, HPV6bE7, HPV11E6, HPV11E7 gene were amplified with PCR using plasmid pUC19/HPV 6b/11 as template and inserted into eukaryotic vector pcDNA3.1-GFP to form recombinant pcDNA3.1-GFP-HPV6b/ll E6/E7, respectively. CRT 180 or CRT 120 DNA fragment was amplified by RT-PCR using total RNA extracted from the excision samples of muscular tissue of human. CRT180/120 and HPV6bE7 fragments were inserted into pcDNA3.1-GFP to form pcDNA3.1-CRT180/ HPV6bE7 or pcDNA3.1-CRT120/HPV6bE7, respectively. The constructed plasmids were identified by enzyme digestion and were further confirmed by DNA sequencing. To establish a HPV6bE7-expressing cell line, the B16 melanoma cell line was transfected with pcDNA3.1-GFP-HPV6b/ll E6/E7 by using the Lipofectamine kit. By the selection of medium containing G418, the G418-resistant positive cell clones expressing HPV6bE7 stably were selected under the fluorescent microscope and identified using RT-PCR.As shown in the study, the established plasmids were digested by enzyme and expected fragments were identified, respectively. After transfection with HPV6bE7 plasmid in B16 cell, green fluorescence located in the cellular nucleus and plasma was observed in positive clones. HPV6b/ll E6/E7 mRNA was also detected by RT-PCR. The results showed that the established cell line positive for HPV6b/l 1 E6/E7 could further be used as a cell line expressing target gene.2.2 The regulatory effect on T lymphocytes and NK cells in mice by DNA vaccineRecombinant DNA and vacant vector pcDNA3.1 were extracted and purified for vaccination by using a plasmid extraction kit. C57BL/6 female 6-wk-old mice, 18-20g, 9 per group, were vaccinated with lOOug of CRT180/HPV6bE7, CRT120/HPV6bE7, CRT180, CRT120, HPV6bE7, vacant plasmid or lOOul PBS via intramuscular injection in the right leg. Mice received two boosts with the same regimen 1 and 2 weeks later. Five days after the last vaccination, the mice were sacrificed and their peripheral blood, spleen and lymph nodes were harvested. Each sample of peripheral blood of vaccinated mice was labeled with CD3/CD4 Ab, CD3/CD8 Ab TCRy 8 Ab and NK1.1 Ab respectively and was analyzed by flow cytometric analysis.There were no significant changes of CD4+ T cell and NK1.1 cells ratios between CRT/HPV6bE7, CRT, and HPV6bE7 vaccination groups. However, CD8+ T cell ratios increased greatly and were remarkably higher in the CRT180/HPV6bE7 or CRT120/HPV6bE7 vaccination group than by using HPV6bE7, vacant vector or PBS (PO.05). Likewise, mice vaccinated with CRT180/HPV6bE7 or CRT120/HPV6bE7 showed a significant increase of TCR y 8 T cell ratios as compared with that of HPV6bE7, vacant vector or PBS (PO.05). Whereas vaccination with HPV6bE7 did not increase CD8+- and TCR y 8 T cells as compared to other control groups. Moreover, there were no significant differences regarding CD8+- and TCRy 8 T cells in mice treated with either CRT180/HPV6bE7 or CRT120/HPV6bE7 DNA. The increase of CD8+- and TCRy8T cells in mice vaccinated with CRT180/HPV6bE7 or CRT120/HPV6bE7 imply that the fusion of CRT180 or CRT120 to HPV6bE7 was important for up-regulating the ratios of CD8+ T cells or TCRy 8 Tin peripheral T lymphocytes, which play an important role since CD8+ T-cell-mediated cytotoxic activity is reported to be critical in controlling HPV infection2.3 The changes of cytotoxic T lymphocyte activity in vitroWe performed a CTL assay using monocytes isolated from spleen and lymph nodes of mice vaccinated with DNA plasmid as effector cells and HPV6bE7-expressing cells as targets cells. Target cells were incubated with effector cells at various effector-to-target cell (E/T) ratios of 1:1, 10:1, 20:1 and a positive or a negative control was used simultaneously. After 6 hours of co-incubation, the centrifuged supernatant was collected to evaluate the lytic activity of T cells by quantitative measuring the amount of lactate dehydrogenase (LDH). The results showed lymphocytes in mice vaccinated with CRT180/HPV6bE7 or CRT120/HPV6bE7 increased specific cytolytic activity at an E/T ratio of 20:1 significantly as compared to that of HPV6bE7, CRT180, CRT120, no insert, or PBS (PO.05). However, no statistically significant difference in cytolytic activity was found between CRT180/HPV6bE7 and CRT120/HPV6bE7 groups at 20:1 E/T ratio. The differences between all these groups at 10:1 or 1:1 E/T ratios did not reach overall statistical significances.Lymphocytes as effector cells adhering to HPV6bE7-expressing cells as target cells at 20:1 E/T ratio and the specific cytolysis with nucleus dust of target cells could be detected in CRT180/HPV6bE7 and CRT120/HPV6bE7 vaccination group by morphologic observation under a microscope. In contrast, the cytolytic phenomena were not obvious in the HPV6bE7 DNA vaccination group at the same E/T ratio.2.4 The changes of IL-2 and IFN-y secretion levelsThe supernatants of mixed cells effector cells and target cells (E/T=20:l) were collected after co-incubation and the concentrations of IL-2 and IFN-y were measured by a direct enzyme linked immunosorbent assay (ELISA). Concentration of cytokines was determined by comparison to a standard curve of serially diluted positive control samples. Both CRT180/HPV6bE7 and CRT120/HPV6bE7vaccinated groups produced significantly higher levels of IL-2 than that of HPV6bE7 vaccinated groups (P<0.05). Similarly, CRT180/HPV6bE7 or CRT120/HPV6bE7 DNA constructs strongly activated IFN-y secretion. IFN-y levels were remarkably lower in HPV6bE7-vaccinated group as compared to CRT180/HPV6bE7 or CRT120/HPV6bE7 vaccinated group (PO.05), but higher than that of CRT180, CRT120, and PBS (PO.05). No significant difference was found between CRT180/ HPV6bE7 and CRT120/HPV6bE7 groups regarding IL-2 and IFN-y secretion. It indicated that vaccination with CRT180/HPV6bE7 or CRT120/HPV6bE7 DNA * plasmids could up-regulate cytokines secretion and increase specific immune responses. 2.5 Anti-angiogenesis activity in vivo.To evaluate inhibitory effect on angiogenesis of generated vaccines in vivo, we employed a bFGF-induced angiogenesis assay using the Matrigel kit. C57BL/6, female, 16-18g, 6-8wk-old mice, 3 per group, were immunized with 100n.g of CRT180/HPV6bE7, CRT120/HPV6bE7, CRT180, CRT120, HPV6bE7, vacant plasmid or lOOjal PBS, respectively, and received a booster application with the same dose 7 days later. A total of 500ul Matrigel mixed with heparin and bFGF was injected subcutaneously in the midabdominal region of DNA-vaccinated mice on the day of last vaccination. After 9d, mice were sacrificed and the Matrigel plugs were removed, fixed in 10% neutral buffered formalin solution, embedded in paraffin, and cut into slices. Tissue specimens were stained with hematoxylin and eosin (HE) or Masson's trichrome for quantitation of microvessel density (MVD). The mean MVD of Matrigel plugs in CRT180/HPV6bE7 or CRT180 DNA-vaccinated groups were significantly lower than that of CRT120/HPV6bE7, CRT120, HPV6bE7, vacant plasmid and PBS-treated mice (P<0.05). Moreover, there was no statistically significant difference between CRT180/HPV6bE7 and CRT180 groups, indicating that CRT180/HPV6bE7 and CRT 180 DNA constructs inhibited bFGF-inducedneovascularization to a similar degree.2.6 Inhibitory effect of vaccine in tumor-bearing mice assayTo further investigate whether the treatment with CRT/HPV6bE7 constructs could generate a potential antitumor effect in vivo, we measured the mean weights, sizes and tumor appearance time in mice treated with HPV6bE7-expressing cells.Tumor treatment experiment: Female C57BL/6 mice, 16-20g, 6 per group were challenged with lxlO5 HPV6bE7-expressing cells by subcutaneous injection in the right back. On the same day the mice were administered with 100u.g of CRT180/HPV6bE7, CRT120/HPV6bE7, CRT180, CRT120, HPV6bE7, vacant plasmid or lOOul PBS, respectively. The mice were boosted by using the same regimen every 7 days for 2 weeks. Five days after the last administration, tumor-bearing mice were sacrificed. The tumors of each group were resected, weighed, fixed in 10% neutral buffered formalin solution, embedded in paraffin and sectioned. Slides were stained with HE and morphology assessed under a microscope. Our data show mean weights of tumors in HPV6bE7 group, CRT 120 group and CRT120/HPV6bE7 group were greater than that of CRT180/HPV6bE7 or CRT180 vaccination group (P<0.01). The histological examination of tumor samples was indistinguishable between mice treated with various DNA constructs with respect to the morphology of tumor cells.Tumor protection experiment: mice were immunized with 100ja.g per mouse of the various DNA constructs as indicated above and boosted 2 times at one-week intervals with the same dose used in the first vaccination. Five days later each mouse was challenged with lxlO5 B16/HPV6bE7 cells by injecting subcutaneously into right back. Tumors growth was monitored every two days for evidence of tumor appearance and size. The tumor-bearing mice were sacrificed 24 days after tumor challenge and tumors were removed for measurement of weights. We did notobserve serious side effects of DNA administration at a dosage of lOOjxg during follow up, indicating that vaccination with the DNA construct is also safe in mice.In the experiment evaluating protective effects, we tested DNA constructs for their ability to delay E7-expressing tumor appearance time and reduce tumors size. All mice receiving no insert, PBS, 6E7, or CRT120 developed tumors within lOd after B16/HPV6bE7 cells challenge. Mice receiving CRT180 only slightly delayed the tumors appearance until day 12. 100% of CRT120/HPV6bE7 treated-mice showed tumor development before day 18. By contrast, all CRT180/HPV6bE7 mice remained tumor-free until day 22 after cells challenge and only 33.3% mice injected with CRT180/6E7 began to develop tumors on day 24. Interestingly, CRT120/E7 delayed tumors formation to a certain degree compared with CRT 120, 6E7, no insert or PBS group, but didn't reduce the rate of tumor growth once tumor came into being compared with CRT180/E7. The tumor growth rates in mice treated with CRT 180 or CRT180/6E7 appeared to be reduced compared to other groups. Also, we found the mean size and weight of tumors in CRT180/6E7-treated group was smaller than those of tumors from mice treated with other constructs (P<0.01). It showed CRT180/HPV6bE7 applied before tumor inoculation could generate a best protective effect against subcutaneous 6E7-expressing tumors among all DNA constructs. Similar anti-angiogenic effect induced by CRT180/HPV6bE7 and CRT180 suggests, at least in part, that aa 120-180 of the CRT molecule are responsible for the anti-angiogenic activity.In conclusion, our data demonstrate that vaccination with CRT180/HPV6bE7 DNA could elicit an efficient E7-specific T cell response and anti-angiogenic effects among all the established DNA vaccines tested. Therefore, CRT180/HPV6bE7 DNA vaccine might be a promising novel therapeutic approach to control HPV6b-associated tumor formation and to reduce or prevent CA.Section three Selective silencing of viral gene HPV6bE6 by specific siRNARNA interference (RNAi) is the process by which small interfering RNA (siRNA) targets sequence-specific degradation of messenger RNA in animal and plant cells. E6 is necessary for completion in early replication cycle of HPV and HPV E6-specific targeting is a rational strategy for gene-specific therapy to eliminate HPV-associated diseases specifically by RNAi. The vulnerability of a certain viral gene to RNA silencing is unknown and the selection of suitable target site is crucial in RNA interfering research. The objection of our study was to investigate if RNAi-mediated gene silencing can be employed to down-regulate exogenous viral gene by using established HPV6bE6-transformed cell as a model system in this current study. This study utilized multiple approaches of RNAi technology, including chemically synthesized siRNAs, the U6 promoter-controlled expression of shRNAs in a GFP-fused form to target the exogenous HPV6bE6 to evaluate the effectiveness of different RNAi approaches on gene silencing. 3.1 The gene silencing of siRNA and shRNA in vitroThe effectiveness of various siRNAs corresponding to HPV 6bE6 remains undefined. It is important to select suitable target region, which depends on proof by assays. The established cell lines positive for target gene HPV6bE6 could be served as an ideal target cell model in RNA interfering experiments. To test the ability of shRNA transcribed from the plasmid vector to inhibit target gene, we transfected the DNA vector into HPV-positive cells.Four pairs of 21-nucleotide duplex siRNA and three plasmid shRNA containing 19-base pair region of HPV6bE6 with GFP as reporter gene, targeting various regions of the HPV6bE6 gene, were devised and synthesized to compare their potential efficacies. Real time-PCR was employed to detect the inhibition rates of target gene by comparing the levels of HPV6bE6 mRNA concentrations of treatedcell line and those of no-treated cell line after transfection. The transfection efficiency of shRNA detected by green fluorescence under microscopic observation 24 hours after transfection was approximately 60-70%. Approximately 70-90% reduction in target mRNA was observed following treatment with both 50nM siRNA-HPV6bE6 and shRNA-HPV6bE6, whereas the cell line without transfection showed no obvious changes of HPV6bE6 mRNA. The results of real-time PCR indicated that both shRNA transcribed from DNA vectors and siRNA specially directed against for HPV6bE6 could efficiently suppress target gene expression to different degrees at post-transcriptional level after entry in mammalian cells. Among four siRNAs, one termed as siB could induce the strongest inhibition of HPV6bE6 mRNA, which targets region 286-304bp in HPV6bE6 ORF. The data showed that the middle region of E6 targeting might induce the more efficient silencing than other regions, including the starting region or ending region targeting. The results indicated that this range targeting induced the comparably stronger silencing. Our results represent a strategy in devising and inducing efficient RNA interference using specific siRNA.3.2 Evaluation of dose-dependent effect and the time-dependent effect of RNA interferenceThe data showed a minimal concentration of siB as low as 1 nM could trigger substantially decrease of homogeneous target gene by amplificative mechanism. siRNA at lnM to 150nM (0.01^g/ml-2ug/ml) could induced the HPV6bE6 mRNA reduction by 70%. It also showed the concentration of siB ranging from In M to 50 nM tend to induce stable gene silence, indicating the inhibition rate could not increased significantly when the concentrations of siRNA increased.HPV6bE6-positive cells were harvested at different treatment time points after transfection. The results showed that the inhibition rates of mRNA levels ofHPV6bE6 at 24h, 48h, 72h, and 96h time points of siRNA treatment were 93.6%, 75.6%, 50.2%, 49.6%, respectively. Our results also showed that the degradation of HPV6bE6 mRNA was maximal at 24h with more than 90% reduction of target gene expression after treatment with siB. It revealed that HPV6bE6 mRNA was suppressed to equal levels whether siB was added 48h or 96h after siRNA transfection. Effective inhibition of HPV6bE6 mRNA was sustained at least over a period of 4 days after transfection with siB. It demonstrated the inhibitory effect of siRNA was stable after transfection into mammalian cells. 3.3 The safty of treatment with siRNAAn ideal therapeutic option should selective and efficient destruction the target without impairing normal tissue. To ascertain if siRNA specific for HPV6bE6 sequences influence the genome of biology, we compared murine GAPDH levels as internal control in cell line with or without transfection. Real Time-PCR revealed that no obvious alteration of GAPDH level of HPV6bE6-positive cells treated with or without siRNA provided evidence for specificity of the RNAi response by specific siRNA. The target gene HPV6bE6 in B16/HPV6bE6 cells showed no marked alteration after treated with shRNA negative control. To investigate the potential nonspecific siRNA effects, we counted the HPV6bE6-cells with or without treatment of four pairs of siRNA. Results also showed that there were no significant cells death observed and no striking morphological differences between siRNA-treated cells and control cells under microscopic observation after treatment with siRNA-HPV6bE6. Also, the growth curve of cell lines showed consistent in treated group and no-treated group. The histological examination of tumor samples showed indistinguishable changes between mice with and without treatment of siRNA with respect of the morphology of tumor cells. It suggests treatment with siRNA could inhibit the accumulation of exogenous viral RNAs but not themechanisms that are critical to cell growth cycle in our system. 3.4 The effect of siRNA-HPV6bE6 in vivoTo assess whether administration of a plasmid DNA vector, from which shRNA can be transcribed and processed into shRNAs or synthetic siRNA could effectively inhibit the target gene expression in vivo, we used an in vivo model expressing HPV6bE6 to test the gene-silencing efficiency of our RNAi method. After ten days the tumor-bearing mice were administered with siB-Oligofectamine mixture in the formed tumors. Mice injected with either synthetic siRNA or plasmid shRNA displayed a significant down-regulation of an exogenous target gene HPV6bE6 expression by more than 40-60% reduction compared with control siRNA in formed tumor expressing HPV6bE6 in vivo. In general, both synthetic siRNAs and plasmid shRNA were well tolerated in mice. These results suggested that plasmid shRNA expressed from a plasmid vector can be processed into siRNA to inhibit target gene production in vivo and in vitro. The down-regulation of target gene HPV6bE6 mRNA could be significantly induced by approach of tumor injection, whereas the target gene showed no marked reduction by vein injection. Also, the system of our animal assays by different injection approach needed to be enhanced.In summary, our data revealed that HPV6bE6 was amenable to siRNA-mediated gene silencing in mammalian cells. The down regulation of E6 is beneficial to accumulation of p53, a key tumor suppressor and cell cycle inhibitor. We found that both vector-borne and synthetic siRNAs specifically directed against the HPV6bE6 gene in HPV-positive cancer cells. Therefore, siRNA-HPV6bE6 provides a rational approach towards the development of novel anti-HPV therapies and itself might be developed as a promising therapeutic agent to counter HPV infectionsConclusion:1. PCR amplification by general primers GP5+/6+ and type-specific primers can offer a feasible approach for the detection and typing of HPV infection of CA clinical patients.2. We have successfully established B16 cell lines expressing HPV6bE7 by transfection with pcDNA3.1-HPV6b/ll E6/E7 plasmid, to serve as target cell models in subsequent experiments.3. We constructed plasmids encoding CRT180 or CRT120 linked with HPV 6bE7 and investigated their activity to modulate immune responses and anti-angiogenic effects both in vitro and in vivo.4. Vaccination with CRT180/6bE7 or CRT120/6bE7 enhanced the presence of CD8+ T cells and TCR y/8 T cells in peripheral T lymphocytes, increased the specific lysis activity against E7-expressing cells and secretion levels of IL-2 and IFN- y by activated T cells in vitro.5. Recombinant CRT180/HPV6bE7 and CRT 180 showed strong anti-angiogenic effects in bFGF-induced angiogenesis in vivo.6. CRT180/HPV6bE7 and CRT180 could significantly inhibit the tumor growth in < tumor treatment experiment and CRT180/HPV6bE7 has superiority to othervaccines in delaying the formation time and size and weight of tumor in tumor protection experiment.7. Recombinant CRT180/HPV6bE7 DNA could elicit an most efficient E7-specific T cell response, anti-angiogenic effects and inhibition of tumor growth among all the established DNA vaccines tested.8. CRT180/HPV6bE7 or CRT180 showed displayed remarkable superiority over CRT120/HPV6bE7 or CRT120in antiangiogenesis in vivo, implying CRT aal20-180 are responsible for the anti-angiogenic activity.9. Both synthetic siRNA and plasmid shRNA targeting the encoded sequences ofHPV6bE6 could inhibit the exogenous viral genes expression in mammalian cells but not the mechanisms that are critical to cell growth cycle.10. Sequence-specific siRNA was capable of inducing selective and efficient silencing of exogenous HPV6bE6 genes and effective inhibition of HPV6bE6 mRNA was sustained at least 4 days.11. Synthetic siRNA-HPV6bE6 or plasmid shRNA-HPV6bE6 could significantly suppress the target gene by approach of tumor injection, which is more efficient than approach of vein injection. |
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