| Condyloma Accuminatum(CA) was one of the most common sexually transmitted diseases(STD)with a high prevalence rate of (31/1,000,000) and has had an increased incidence in our country recently. Human Papilloma virus type 11(HPV11) and 6(HPV6) were the primary viruses that caused Condyloma Accuminatum , which accounted for 95% of all pathogens that caused CA pathological changes. Although a large number of study had been performed to investigate the pathogenesis and the preventive and curative measures of Condyloma Accuminatum, because of the pathogenesis of CA being still unkown, there had been no substantial progress to achieve. Until now, there have been no efficient curative measures to eliminate the viruses in patients body completely. So, CA was susceptible to recurrent attacks and could not be cured in no time. The immune state of patients determined the natural history of HPV infection and the outcome of related diseases that HPV11 causeed. The investigators thought highly of the disorder of immunological function of patients with CA. Efficient immune response could not be established, which was the primary reasons of the HPV infection repeatedly. Many experts believed that using the immunomodulator could modulate specifically the immune system of anti-HPV and decrease the recurrence rate of the CA after the removal of wart. 90% patients infected with HPV could eliminate the viruses naturally. However, the factors that determined whether the viruses were eliminated or remained persistently or lesion deteriorated were indefinite. Therefore, there were important theoretical significance and clinical value to explore the immunological pathogenesis of CA. Toll-like receptors (TLRs) were the new pattern recognition receptors that were involved with recognition of microbial pathogens and played an important roles in linking the innate and required immunological response in the mammalian species. So far, there are eleven TLRs that have been found and different TLRs recognize different microorganism components. Among them, TLR9 recognizes the unmethylated CpG motifs-containing synthetic ODNs and DNA from bacteria or viruses and activates the following effectors IL-1/Toll-like receptor associated kines(IRAK), tumour necrosis factor receptor associated factor6(TRAF6)and NF-kappaB by turns and lead to the secretion of Th1 type cytokines TNF-alpha, IL-12 and IFN-alpha, which play important roles in anti-infection, anti-tumor, mediating apoptosis and autoimmune disorder. In our study, the eukaryotic expression plasmid of pCIneo-hTLR9 were constructed and expressed on HEK293 cells and the investigation of the relation between TLR9 and HPV11 and the immunologically molecular mechanism of infection by HPV11 were performed. The following three experiments were performed in this study. Part I. Construction of recombinant plasmid pCIneo-hTLR9 and expression on HEK293 cells. The recombinant plasmid pCIneo-hTLR9 were constructed with directional cloning technique and transfected into HEK293 cells. Obstaining successfully the eukaryotic expression plasmid pCIneo-hTLR9 was to provide prerequisite tools and experimental basis for the next study. Part II. Analysis and identification the methylation state of CpG motifs in HPV11 DNA. Counting the total number, classification and localization of CpG motifs in HPV11 DNA digested from the recombinant plasmid pBR322-HPV11 were performed with Computer-assisted . We identified the methylation State of CpG motifs in HPV11 DNA through analysis with Restriction Endonuclease Polymerase Chain Reaction(PCR)。That's to say, using restriction endonucleases Acc II, Hae II and Hpa II to digest the HPV11 total DNA, respectively. Then amplification the genes of interest due to PCR by the flanked sequence of Hpa II, Acc II, Hae II digest sites as primers and Hpa II digested products as template. It found 155 CpG motifs and 14 sequences with non-methylated CpG motifs flanked by two 5'purine and 3'pyimidines, such as GACGTT, GACGCC, GGCGCC,AACGCC AGCGTT and so on. Part III. The molecular mechanism by TLR9-mediated immunological recognition. The HPV11 DNA were used to treat the HEK293 cells expressed pCIneo-hTLR9 (HEK293+), Reclamation the supernatant and measuring the concentration of IFN-alpha, IL-12 and TNF-alpha by relative ELISA kit after incubation for 20h. Chloroquine(CQ)interference experiment was performed to study the signal of TLR9-HPV11 DNA. Results showed TLR9 was required for HPV11 DNA inducing the secretion of IFN-alpha, TNF-alpha and IL-12 from HEK293+, HPV11 DNA provided the ligands for TLR9 expressed on HEK293+ and CQ could inhibit the secretion of TNF-alpha, IFN-alpha and IL-12 from HEK293+ cells inducing by HPV11 DNA. The treatment by HPV11 DNA could increase the expression of hTLR9 protein in HEK293+ cells. |
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