Construction Of Recombinant Adenovirus With Human Papillomavirus 16 E6/E7 Gene And Its Effects On CASKI Cell In Vitro

Cervical cancer is one of the commonest gynecologic malignance in the world with approximately 470,000 new cases and 23,000 deaths of cervical cancer occur each year, and is becoming younger. Although surgery, radiotherapy and chemotherapy represent an effective modalities of treatment for cervical cancer, some of these patients overall will still develop recurrent or metastatic disease. Thus, more and more people have a notion that the study of cancer should begin in their biology and master the tumour characteristic to study intensively the biotherapy, especially the tumour immunotherapy.Dendritic cell have been identified as the most effective antigen presenting cell, which can induce cytotoxic T lymphocytes, play an important role in therapeutic antitumour immunity.Our study was to use the recombinant adenoviruses carrying human papillomavirus 16 (HPV-16) E6/E7(pAd-E6/E7) to infect mouse immature dendritic cell, to observe the lethal effect and apoptosis to CaSki cells of specific cytotoxic T lymphocytes (CTLs) triggered by DC vaccine in vitro. We want to provide evidence for studying of the immunotherapy of cervical cancer. The result are summarized as following 2 parts.(1) The preparation of DC vaccine: The bone marrow cells were isolated and then cultured with rmGM-CSF and rmIL-4. Large numbers of colonies and half-adherent imDC could be observed by contrast phase microscope. CD11_C , the relative specificity surface expression of imDC was 71.19% ,is high level expression while other relative specificity surface expression including CD₄₀, CD₈₆ and MHCâ…¡were 34.5%,39.53% and 55.35% were low level expression examined by FCM which were consistent with the characteristic surface expression of imDC. After being infected with pAd-E6/E7 24h, different sizes of dendritic and burr-like protrusions DC could be seen by contrast phase microscope. Some Green fluorescence, and some dendritic processes appear could also be seen by confocal microscopy. CD11_C, CD₄₀, CD₈₆ and MHCâ…¡which are the relative specificity surface expression of DC were 62.87%,55.5%,78.51%,95.95%, consistent with the characteristic surface expression of mDC. E6 protein expressed in dendritic cell vaccines which was examined by Western blot analysis .(2) Dendritic cell vaccine inducing specitific CTLs to kill CaSki cells: DC vaccine was used to induce specific CTLs, and after co-culturing the CTLs with CaSki cells, and CTLs were washing-out softly by PBS. The apoptosis of CaSki cells were detected by TUNEL and approximately 72.82% apoptosis of CaSki cells were could been examined by FCM, while there were no significant change in the control groups.In order to compare the gene-modified DC vaccines and the loaded with tumor cell lysates of DC vaccine which is good at inducing CTLs to kill tumor cells, the experiment used pAd-E6/E7 infected DC vaccine and loaded-CaSki cells lysates DC vaccine to induce specific CTLs, and we discovered that the former DC vaccine was better than the last one to kill CaSki cells which was examined by CCK8.The pAd-E6/E7 infected DC vaccine has a good prospects for clinical application.