The Molecular Biologic Carcinogenesis Of Human Papillomavirus In Cervical Cancer And The Investigation Of Virus Like Particle As Vaccine To Protect Women From Human Papillomavirus Infection

Objective To construct the Baculovirus expression vector for human papillomavirus type 16 L1 gene and transform the Bacmid to insect sf9 cell line to express HPV-16 L1 virus like particals. Methods The HPV-16 L1 gene was amplified by PCR and cloned into plasmid pFast HTb , and the combinant plasmid pFast HTb-HPV16 L1 was transformed to competent cells DH10Bac. A transformant containing the target gene was obtained and named as Bacmid-HPV16 L1. All the cloning steps were electrophoresed in EB stained 1% agarose gel. After transfecting this transformant into Sf9 cells , its expression in Sf9 cells was detected by SDS-PAGE and electron microscopy.Results The experimental results showed that the recombinant buculovirus was obtained and the transformant Bacmid-HPV16 L1 virus like particals were expressed in insect Sf9 cells. This recombinant transformant can be used as the protein accessed high biological characteristics for the functional study on Bacmid-HPV16 L1 virus like particals. Objective To found the methods of isolation, cultivation, and identification of human corneal fibroblasts in vitro. Methods The human cornea fibroblasts grew under the anchoring tissue and were cultured in stable condition for future generations. Cellular morphologies were observed by inverted phase contrast microscopy and electron microscope and the proliferation characteristic were described by growth curve and flow cytometry. The cultured cells were identified by immunostaining of vimentin, collagenase type I and keratin and chromosome analysis. Results The isolated fibroblasts could grow and proliferate in vitro showing typical characteristic of fibroblasts. The immunostaining of vimentin was positive and collagenase type I and keratin were negative in cultured fibroblasts and the number of chromosome was 46. Conclusions Acquired human cornea fibroblasts can be cultured in stable condition in vitro, and sufficient and reliable cells can be obtained for the study of the mechanisms of tumor. To investigate the fuctions of HPV 16 E6E7 protein in the development of cervical cancer, a recombant plasmid pEGFP-HPV16E6E7, containing green fluorescent protein and E6E7 protein was constructed and was transfected into HPV negetive cell line C33A and primarily cultured human fibroblast (HFB) mediated by liposome. The efficiency of tranfection was determined by inverted fluorescenct microscopy and the expression of E6E7 were detected by western blot. It showed that transfection of HPV-16 E6E7 gene developed two pairs of transfected cell types to study the carcinogenesis of E6 and E7 protein. Objective To detect the expression of Angiogenesis-associated factors in two pairs of human papillomavirus type 16 E6E7 protein positive and negative cells and to explore their possible roles in tumor metastasis and progression of E6E7protein.