| Bcl-2 family is one of the most important genes families to regulate apoptosis. Bcl-2 family contain anti-apoptotic family members (e.g. Bcl-XL,Bcl-2), and pro-apoptotic family members, such as Bik and Bax. The abnormality of Bcl-2 family not only relates to the genesis and progression of tumors, it also links to the chemosensitivity of some anticancer drugs. The low expression of pro-apoptotic family members or the over expression of anti-apoptotic family members in many lymphoma cells result in the insensitivity of inducing apoptosis as well as the resistance of chemotherapy for lymphoma cells. Bik is one of the most potent BH3-only proteins, shown to interact with more anti-apoptotic family members and have stronger binging force with widely studied Bcl-2 than others pro-apoptotic family members. Bik mutant (BikDD), in which changes were made in T33D and S35D to mimic phosphorylation at these two residues, can enhance its binding affinity with the antiapoptotic proteins Bcl-X1 and Bcl-2 and show more potent than wild-type bik in inducing apoptosis and inhibiting cell proliferation of cancer cells. Transduction of BikDD gene into lymphoma cells may be a promising method of gene therapy for lymphoma. One of the major obstacles of gene therapy is that the ectopic gene cannot express highly in targeted cells. The objective of this study focuses on how to guarantee BikDD gene expresses highly in lymphoma cell lines and research the apoptotic effects of BikDD on lymphoma cell lines.In the first part of this study, we generated luciferase vectors contain Survivin promoter, hTERT promoter and CMV promoter respectively. We detected the luciferase expression of vectors in different lymphoma cell lines and hepatic cell line chang liver to evaluate the efficacy and lymphoma specificity of promoters.In the second part, we constructed three plasmids contained different elements of WPRE, TSTA and VISA under control of Survivin promoter. We detected the luciferase expression of vectors in different lymphoma cell lines and hepatic cell line to select the most efficient amplification system, which would be used to construct high efficient and lymphoma-specific gene expression vectors with Survivin promoter.In the third part, the lymphoma cell lines Raji, Ramos, U937, hut78 and hepatic cell line chang liver were transfected with the recombinant BikDD vector, which contained a 987 bp human Survivin promoter, a mutant Bik cDNA, as a pro-apoptotic gene, and VISA signal amplification system. Then, Western blot was used to assess the different lymphoma cell lines expression of Bik. We also used the MTT assay and flow cytometry to detect growth inhibition and pro-apoptotic effects on lymphoma cell lines.Results:The luciferase detection results showed that Survivin promoter was more efficient and lymphoma-specific than hTERT promoter. S-VISA and S-TSTA showed more efficient than pGL3-CMV in lymphoma cell lines Raji, Ramos, Hut78 and U937 (p<0.05), but less efficient than pGL3-CMV in hepatic cell line chang liver. S-VISA was selected as highly efficient and targeted gene expression vector in lymphoma cell lines for its higher efficacy in Raji, Ramos and U937 than S-TSTA (p<0.05)Results of Western blot showed that Bik expression of lymphoma cells was lower than Bik expression in chang liver cells in different degree, and Bik expression increased in different degree after lymphoma cells were transfected with S-VISA-BikDD plasmid. S-VISA-BikDD resulted in significant growth inhibition in all transfected cell lines except chang liver. S-VISA-BikDD resulted in significant apoptosis in transfected Raji cell line.Conclusion:So far as lymphoma cells and hepatic cells, the Survivin promoter can make Bik gene expressing targeted in lymphoma cells. VISA system amplifies the activity of Survivin promoter effectively. S-VISA-BikDD pro-apoptotic vector can lead the lymphoma cells to apoptosis and growth inhibition. S-VISA-BikDD is an effective pro-apoptotic vector for gene therapy in lymphoma.
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