Exploring In Vitro Pharmacodynamical Models For Drugs Against Human Papillomaviruses

Human papillomaviruses (HPVs) are small, double-stranded DNA viruses that infect mucosal and cutaneous epithelia, and cause benign or malignant neoplastic lesions. More than 100 HPV genotypes have been identified and one-third of them infect anogenital region, which can be divided into high-risk and low-risk types depending on their association with malignancy. The high-risk HPVs, such as HPV16 and HPV-18, are the etiological agents of cervical cancer and have been extensively studied. The low-risk HPVs, such as HPV-6 and HPV-11, are generally associated with anogenital and laryngeal warts, and also important types due to their increasing incidences.The life cycle of HPVs is strictly dependent on epithelial differentiation and a convenient tissue culture system for propagating the virions is lacking, which has substantially retarded progress in the development of drugs against HPV infection. In this study, we attempted to develop an in vitro experimental system for low-risk HPVs using an immortalized human keratinocyte strain, HaCaT, by transfection or infection method. We also examined several related infectious markers including HPV DNA, mRNA and caspid protein by various techniques. Our studies were divided into two parts as follows.Part I Transfection of HaCaT keratinocyte with HPV-11 genome DNA and detection of infectious markers.Objective To investigative the possibility of preparing HaCaT keratinocyte stably maintaining HPV11 genome by transfection and selection methods; to explore the appropriate infectious markers and efficient methods for theirs.Methods Escherichia coli containing pBR322.HPVll plasmid was cultured and amplified.Then the plasmid was extracted, purified and digested with BamH I enzyme to release viral genome from the bacterial vector. After recovering from the low-melting-pointing agarose gel by electrophoresis, the genome was self-circulated...