| Cervical cancer is one of the most globally common cancers in women, just second to breast cancer in morbidity. Worldwide, 27 million patients each year die from cervical cancer. Currently, surgery, radiotherapy and chemotherapy still be dominant in cervical cancer treatments, but these therapys also show serious untoward reaction in patients. Study on biologic agent will be imminent in preventing and treating cervical cancer.Epidemiological and virological data show that human papillomavirus(HPV) infection is the major etiological factor of cervical cancer. HPV DNA can be found within 99.7% of the malignant cells of cervical cancers, of which, 57.4% was HPV l6 type. The discovery indicate that cervical cancer vaccines is expected to be the first human cancer vaccines.HPV is a small, nonenveloped virus. The genome consisit a single copy of circular double-stranded DNA including early region (E), late region (L) and upstream regulatory region (URR). E6 and E7 protein of High-risk HPV is viral oncoprotein which can lead to immortalization by interruptting the cell growth-regulatory pathways. L1 major capsid protein of HPV can self-assemble into VLPs (virus-like particles) and show good immunogenicity.The current HPV prophylactic vaccine candidates are based on VLPs. HPV-L1 VLPs are morphologically and antigenically similar to authentic virions without infectious and carcinogenic. However, the VLPs are mainly obtained from insect cells infected by recombinant baculovirus vector or yeast with recombinant vector. The process is cumbersome, and expensive for VLPs application in developing countries.Currently, most of therapeutic HPV vaccines optimize HPV E6 and E7 target antigen to induce cellular immune response and inhibit cancer growth. However, they are potential carcinogens, may lead to cell abnormal proliferation and other adverse reactions. RNA interference is a new gene therapy technology. Some researchers have succeed in silencing HPV-E6 and/or E7 gene of cervical cancer by RNA interference. However, siRNA molecules enter target cells and maintain stability limit its application in vivo.Attenuated Salmonella is the first recombinant bacteria used to delivery antigen for 25 years. More and more evidence support that alive attenuated Salmonella can induce humoral and cellular immune responses in vivo. Attenuated Salmonella can induce mucosal immune response by non-injection at less than 3 times. In tumor tissue, the attenuated Salmonella can make best use of the hypoxic microenvironment of tumor, nutrients and local immunosuppressive microenvironment. It is the biology advantage of the attenuated Salmonella that make it to be a vaccine vector used for the cervical cancer prevention and therapy.The purpose of this study is to construct pcDNA3.1-HPV16-L1-siRNA-E6 plasmid, transform it into attenuated Salmonella, vaccinate mice by intranasal, and observe the prevention and treatment effect of cervical cancer.The result proved, attenuated Salmonella carrying pcDNA3.1-HPV16-L1 induced anti-HPV16-L1 antibodies in mice successfully after vaccinate intranasally. siRNA-E6 designed by the sequence of HPV16-E6E7, and plasmid carrier attenuated Salmonella was innovative applicated in the cervical cancer treatment and result in significant antitumor effect. The recombinant attenuated Salmonella with pcDNA3.1-HPV16-L1-siRNA-E6 plasmid also success contribute to cervical cancer prevention and treatment.1. Attenuated Salmonella carrying pcDNA3.1-HPV16-L1 as DNA vaccineAs the purification process of recombinant protein is complicated, protein vaccine is expensive. Further more, the multivalent protein vaccine is available. One of main purpose of this study is to develop preventive DNA vaccine. In this study, HPV16-L1 gene was amplified from fresh human cervical cancer genom, prokaryotic expression recombinant plasmid pET28a-HPV16-L1 was constructed and transfered into E. coli BL21(DE3) to express HPV16-L1 protein.After denatured and purified, virus-like particles of 50nm diameter could be observed by transmission electron microscope. Anti-HPV16-L1 antibody could be detected in the serum fluid of mice, after mice be vaccined with emulsified VLPs and Freund adjuvant.Further more, the eukaryotic expression plasmid pcDNA3.1-HPV16-L1 was construced and transfected into hamster kidney cell line BHK with liposome. After 48h, target protein was detected. The pcDNA3.1-HPV16-L1 was transformed into attenuated Salmonella Ty21a and PhoP/PhoQ used to vaccined model mice by intranasal immunization. Anti-HPV16-L1 antibodies can be found in serum and vaginal fluid of mice. The antibody levels in serum and vaginal fluid was increased with time, reached a peak in the 15th day and maintain a high level to the end of the experiment. In this study, the serum content of IL-2 and INF-γincreased in the group of attenuated Salmonella carrying pcDNA3.1-HPV16-L1, compared with the control group.2. siRNA-HPV16-E6 inhibit the proliferation of cervical cancerAt present, most of therapeutic HPV vaccine induce cellular immune response by HPV E6 and E7 proteins. E6 and E7 proteins may lead to cell abnormal proliferation for its potential carcinogens. In this study, siRNAs were emploied to silence HPV E6 and E7 oncogene and inhibit the growth of cervical cancer.HPV E6 and E7 gene sequences was used to design siRNA-E6A, siRNA-E6B and siRNA-E7 for constructing pGC-siRNA plasmid. After transfected into cervical cancer Siha cells, pGC-siRNA in siE6A group and siE7 group down-regulat the E6 and E7 transcription and expression, inhibit cell growth, promote cell apoptosis, and up-regulate the p53, bax, caspase-9 and caspase-3 expression.After the cervical cancer model mice was established, pGCsi-E6A plasmid was transfected into tumor tissue. The E6 and E7 expression were dowm-regulated, tumor growth was inhibited, the apoptosis-related genes expression were up-regulated in the experimental group compared with control group. All datum support pGCsi-E6A plasmid has antitumor effect in vivo.3. Cervical cancer prevention and therapy by attenuated Salmonella carrying pcDNA3.1-HPV16-L1-siRNA-E6In this study, pcDNA3.1-HPV16-L1-siRNA-E6 was constructed for the first time, transformed into attenuated Salmonella PhoP/PhoQ to observe the treatment effect of tumor model mice. RT-PCR, western blotting and immunohistochemistry assay showed that L1 expression is upregulated in tumor tissues, whereas E6 and E7 expression are downregulated. The results indicating that attenuated Salmonella could carry the plasmid to tumor tissue and realize HPV16-L1 protein expression and E6E7 gene silenc successfully. Attenuated Salmonella with empty vector, pGC-siE6, pcDNA3.1-HPV16-L1, and pcDNA3.1-HPV16-L1-siRNA-E6 can inhibit tumor growth compared with the control group, suggesting that attenuated Salmonella has antitumor effect. Compared with pcDNA3.1-HPV16-L1 and pGC-siE6, co-expression plasmid can further reduce tumor size, suggesting that HPV16-L1 expression and E6E7 silencing in tumor have synergistic effect by immune regulation and RNA interference in cervical cancer.prevention and treatment.For the cervical cancer prevention and therapy, this study will contribute to a new productive thinking. |
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