The Effects Of Anti-HPV16E6-ribozyme On Activity Of Telomerase,Radiation And Chemotherapy Sensitivity Of Cervical Cancer Cell Line

BackgroundThe human papillomavirus (HPVs) are small DNA tumor virus that infect epithelial cells and induce proliferative lesions. Substantial epidemiologic date along with in vitro and in vivo studies have led to the implication of particular HPVs with the development of epithelial malignancies. Greater than 90% of all cervical carcimomas are positive for HPV infection. Most of these lesions are caused by infection with mucosal-associated high-risk HPV subtypes. HPV are the most important etiologic factor for cervical carcinoma. E6 virus genes is one of the most important oncogen and Levels of expression of E6 transforming oncoproteins of high risk HPV genotypes,such as HPV 16,appear to be necessary for maintaining the malignant phenotype. Gene therapy is one of the most promising approaches to date for treating several inherited and acquired diseases,such as cancer,neurological,cardiovascular and viral diseases. Recently,a new class of genetic tools,called ribozymes,has been identified,capable of controlling gene expression through the manipulation of RNA. Ribozymes are catalytic RNA molecules,which can be designed to bind RNA in specific manner like an antisense. Additionally they possess catalytic properties,which enable them to cleave or edit RNA molecules. Radiation treatment represents a standardized and effective modality for contemporary cervical carcinoma therapy. Chemotherapy,which severs as an adjuvant treatment,became more and more important in the treatment of middle and late stage cervical carcinoma . New consideration and methods will be provided to treat cervical carcinoma by combination with gene therapy,radiotherapy and chemotherapy .ObjectiveThe study was intended to investigate the malignant phenotype,theTelomerase activity,radiation sensitizing effect and chemotherapy sensitizing effect of anti-HPV16 E6-ribozyme on cervical carcinoma cell line,and clarified mechanism of gene expression modulation. It's the possibility and practicality of a new therapeutic strategy,ribozyme and combination with ribozyme,chemotherapy and radiotherapy in treatment of cervical cancer was explored in order to enhance curative effect as far as possible.Methodsl.With the method of lipofectin transfection,the anti-HPV16E6-ribozyme and empty eucaryotic expressing plasmids were transfected into CaSKi cell,which named as CaSKi-R and CaSKi-P,respectively. The expression of ribozyme in transfected cells was observed by RNA dot blot. The amounts of E6 mRNA in the three kinds of cells were detected by Northern blot. 2.The morphology,and the soft agar forming ability,were studied in CaSKi cell and the transfected cells. The growth rates of the three kinds of cells were examined by cell count. Percent ate of three kinds of cells in different phase of cell cycle determined by flow cytometry.3.Telomerase activity in three kinds of cells were examined by TRAP-ELISA. The expression of protein and gene of p53,bcl-2,c-myc was detected by flow cytometry,western blot and RT-PCR,respectively.4. MTT colorimetric assay was applied to detect X-ray induced damage and cytotoxicity of chemotherapy drug in three kinds of cells in vitro. MTT colorimetric assay and colony formation test were used to examine their sensitivity to radiotherapy and chemotherapy,and effect of the anti-HPV16E6-ribozyme on sensitivity of cervical carcinoma cell line to radiotherapy and chemotherapy . The ability of each cell to form tumor and sensitivity to cisplatin in vivo were studied by analysis of immigration tumor in nude mice.5. The apoptosis rates of each cell was determined by PI/Annexin V stained methods. Agarose gel electrophoresis of DNA from three kinds of cells undergoing X irradiation or treated with DDP was detected. Ultrastructrre of three kinds of cells was also observed by transmission electron microscope treated with irradition or chemotherapy drug.6.Expressions of p53,bcl-2,bax and c-myc proteins in three kinds of cells treated with X-ray or chemotherapy drugs were determined by Flowcytometry analys...