Induction Of TRAIL Eukaryotic Plasmid And Taxol On Apoptosis For Cervical Cancer Therapy

Cervical cancer is a leading cause of cancer-related deaths in China. The incidence and mortality rates of cervical cancer have increased markedly. Women currently diagnosed at the early stages of cervical cancer can, in many cases, be effectively treated by surgery or radiation. At present, the primary difficulty is to cure advanced stage of cervical cancer . As a new treatment of tumor gene therapy or biology therapy is thought highly of increasingly. It is demonstrated that TRAIL can induce apoptosis in a wide range of cancer cells, and the most important is that TRAIL can selectively induce apoptosis in tumor or transformed cells, but not in normal cells. TRAIL is also showed a synergic effect with the traditional radio and chemotherapy. Therefore, it will be a promising candidate for cancer therapy.TRAIL is a characteristic type II transmembrane protein. The extra cellular part of it can be proteolytically cleaved from the cell surface and resulting soluble molecular in which the functional domain is between _114-281 amino acids (sTRAIL).Taxol is a new chemotherapy drug and has distinctive trait of resisting microbule depolymerizing.This study is to explore TRAIL eukaryotic expression plasmid and Taxol for the cervical cancer therapy in vitro and in vivo. 1.Construction of a recombinant plasmid expressing pEGFP-hTRAIL_114-281Objective: To construct fusion gene expression plasmid pEGFP-hTRAIL_114-281. Methods: To constructe fusion gene expression plasmid, TRAIL_114-281gene was amplified from TRAIL full length plasmid by PCR and cloned into the EGFP downstream of the vector pEGFP-C1. Insulin signal peptide was cloned into the EGFP upstream of the vector pEGFP-C1. The recombinant plasmid had been confirmed by restriction enzyme digest and sequence analysis. Result: A507bp was obtained which was identified by enzyme digestion and sequence analysis. Conclusion: The recombinant eukaryotic expression vector for TRAIL_114-281gene had successfully constructed pEGFP-hTRAIL_114-281.2. TRAIL eukaryotic plasmid and Taxol on apoptosis for cervical cancer therapy in vitro:Objective: To explore TRAIL eukaryotic plsmid and Taxol on apoptosis for the therapy of the cervical cancer Hela cell in vitro. Methods: The cervical cancer Hela cell was transfected with plasmids pEGFP-TRAIL_114-281 or pEGFP-C1 plasmid. To detect pEGFP-TRAIL_114-281 and Taxol inhibition Hela cell proliferation by MTT. The cells were also analyzed for cell cycle phase distribution and apoptosis rate by flow cytometry, and stained by Acridine Orange apoptosis detection apoptosis. To determine the expression levels of the DR4 and DR5 by the semi-quantitative RT-PCR analysis.Western blot analyses with the samples extracted from transfected and control cells were performed. Result: (1) The stable cell lines which could maintain TRAIL_114-281 plasmid and pEGFP-C1 plasmid in transfected cells was established.(2) Transfection with pEGFP-TRAIL_114-281 inhibited the growth of Hela cell and induced apoptosis.(3) pEGFP-TRAIL_114-281 plasmid and Taxol showed a synergistic effect for suppression Hela cells. (4) Taxol can increase the expression level of the DR5 of the Hela cell. The results of Western blot analyses showed that TRAIL can increase the Caspase8 expression level and Taxol can increase the Caspase8 and decrease Bcl-2 expression level .Conclusion: TRAIL plasmid may 1.Construction of a recombinant plasmid expressing pEGFP-hTRAIL_114-281 Objective: To construct fusion gene expression plasmid pEGFP-hTRAIL_114-281.Methods: To constructe fusion gene expression plasmid, TRAIL_114-281gene was amplified from TRAIL full length plasmid by PCR and cloned into the EGFP downstream of the vector pEGFP-C1. Insulin signal peptide was cloned into the EGFP upstream of the vector pEGFP-C1. The recombinant plasmid had been confirmed by restriction enzyme digest and sequence analysis. Result: A507bp was obtained which was identified by enzyme digestion and sequence analysis. Conclusion: The recombinant eukaryotic expression vector for TRAIL_114-281gene had successfully constructed pEGFP-hTRAIL_114-281.2. TRAIL eukaryotic plasmid and Taxol on apoptosis for cervical cancer therapy in vitro:Objective: To explore TRAIL eukaryotic plsmid and Taxol on apoptosis for the therapy of the cervical cancer Hela cell in vitro. Methods: The cervical cancer Hela cell was transfected with plasmids pEGFP-TRAIL_114-281 or pEGFP-C1 plasmid. To detect pEGFP-TRAIL_114-281 and Taxol inhibition Hela cell proliferation by MTT. The cells were also analyzed for cell cycle phase distribution and apoptosis rate by flow cytometry, and stained by Acridine Orange apoptosis detection apoptosis. To determine the expression levels of the DR4 and DR5 by the semi-quantitative RT-PCR analysis.Western blot analyses with the samples extracted from transfected and control cells were performed. Result: (1) The stable cell lines which could maintain TRAIL_114-281 plasmid and pEGFP-C1 plasmid in transfected cells was established.(2) Transfection with pEGFP-TRAIL_114-281 inhibited the growth of Hela cell and induced apoptosis.(3) pEGFP-TRAIL_114-281 plasmid and Taxol showed a synergistic effect for suppression Hela cells. (4) Taxol can increase the expression level of the DR5 of the Hela cell. The results of Western blot analyses showed that TRAIL can increase the Caspase8 expression level and Taxol can increase the Caspase8 and decrease Bcl-2 expression level .Conclusion: TRAIL plasmid may...