| Human papillomaviruses are nonenveloped double-stranded DNA tumor viruses.Over 90% of all cervical cancers have been found HPV positive,with particular "high-risk"HPV types(16,18,31,etc)mostly commonly found. The genomic organiziton of HPV have been established. They comprise upstream regulate region(URR), early region and(E) and later region(L). The early region have eight open read frames(ORFs), E6,E7,El,E8,E2,E4,E3,and E5 respectively.The high-risk HPV E6 and E7 oncoprotein exert profound effects on the targeted cells and activate tumor suppressor protein.These viral oncogene are involved in the progression and maintainace of transformation Therefore,to target the high-risk HPV E6 and E7 oncoprotein has important role on treatment of cervical carcinoma.Double-stranded RNA were inheritable substance. They exit in many plants,animals,fungi and viruses.RNA interference(RNAi) could result in sequence-specific gene silencing by exogenous double-stranded RNA. Owing to sequence-specific RNAi could degrade dsRNA-mediated mRNA .Thus RNAi could inhibit the expression of targeted gene.It is similar with gene "knock-out" ,but faster and simpler. RNAi have been discovered in many organisms,such as plants,fungi,drosophila and zebrafish etc.However,it has not been possible to detect potent and specific RNAi in commonly used mammalian cell culture systems applying dsRNA that varies in size between 38 and 1662 base pair (bp). With base-pair overhanging at ends short interference RNA(SiRNA)were double-stranded RNA which shorter than 25bp.These SiRNA could not activate the protein kinase K(PKR) and trigger interferon reactions,therefore could maintain vitality of the mammalian cells.So in mammalian cells, to investigate the function of gene and specific gene silencing by using SiRNA become feasible.Now, gene therapy of cervical cancer is one of the new strategy in the cervix tumor therapy and has been intensely studied. Though the gene therapy, such as antisence therapy and suicide gene therapy, have been made progress.They have many problems The high-risk HPV E6 and EToncoprotein were important oncoprotein of cervical cancer.Can we synthesize SiRNA targeted the high-risk HPV E6 and EToncoprotein by using RNAi and treat cervical cancer?Here,we first measure the expression of HPV18 E6 and E7 in Hela cells by S-P immunohistochemical methods and reverse transcription PCR (RT-PCR). Then we synthesize 21-neuclotide SiRNA targeted HPV 18 E6 and E7.After transfection Hela cells,we measure HPV18 E6 oncoprotein by S-P immunohistochemical methods.These may be provided a new strategy for the prevention and therapy of cervical carcinoma.Part IAn experimental study on the expression of Human Papillomavirus 18 E6/E7 in Hela cervical cancer cellsObjective: Cervical cancer is one of the most common reproductive cancer of women.Infection of high-risk type human papillomavirus is one closely associated with cervical cancer. This study is to measure the expression of HPV18 E6/E7 in Hela and to investigate the significance in cervical carcinoma.Methods: The expression of HPV18 E6 in Hela were investigated by S-P immunohistochemical methods Expressions of HPV18 E6/E7mRNA determined by reverse transcription PCR(RT-PCR).Results: There were expressions of HPV18 E6/E7 mRNA and protein in Hela.Conclusions: There were expressions of HPV18 E6/E7 in Hela and they may be targeted gene of treatmentof cervical carcinoma.Part IISpecific suppression of HPV18 E6 oncoprotein by short interference RNA in Hela cervical carcinoma cellsObjectiverBy RNAi,we investigate whether HPV18 E6 SiRNA targeted Hela cells cervical carcinoma can inhibit the expressions of HPV18 E6 oncoprotein.Methods:For SiRNA studies in mammalian cells,we synthesize HPV18 E6 and E7 SiRNAs in vitro using T7 RNA polymerase.The products were checked by running on 2% agarose gel.Then Hela cells were grown in standard conditions and transfecting with lipofectamine?The cells were divided into four groups,A group:HPV18 E6 SiRNA;B group:HPV1...
|
PDF Full Text Request