Augmentation of neu DNA Vaccine Therapeutic Effect with TSP-1 (Thrombospondin-1) siRNA

• Main Authors: Shih-Shien Huang, 黃仕憲
• Other Authors: Ming-Derg Lai
• Format: Others
• Language: zh-TW
• Published: 2007
• Online Access: http://ndltd.ncl.edu.tw/handle/03921801838821637307

碩士 === 國立成功大學 === 生物化學研究所 === 95 === ABSTRACT The HER-2/neu oncogene is amplified and overexpressed in 20% of primary human breast cancer and due to the tumor specificity of its overexpression. The HER-2/neu oncoprotein is considered one of the most attractive targets for immunotherapeutic intervention. In our previously study, we successfully used xenogeneic DNA vaccine combining variety cytokines against mouse tumor cells overexpressing endogenous p185neu. Until now, we still try to discover the more powerful adjuvant to enhance the therapeutic efficacy of neu DNA vaccine. Thrombospondin-1 (TSP-1) consist of a family of 5 extracellular domains that participate in cell-to-cell and cell-to-matrix communication and it was reported to be an autocrine negative regulator of human DCs and to activate in vivo latent TGF-β. Base on its unusual role in immune response, we constructed the plasmid expressing TSP-1 siRNA and human-cyto-N’-neu, then check the neu expression level and knockdown effect of TSP-1 siRNA by immunohistochemistry and western blotting. The results suggested that vaccinated mice with TSP-1 siRNA by gene gun could inhibit the TSP-1 expression in draining lymph node. We found that the human-cyto-N’-neu-TSP-1 siRNA has better therapeutic effect. Our results provide novel strategies to enhance neu DNA vaccine therapeutic efficacy. We also found that Treg specific marker, foxp3, decreased in splenocytes of human-cyto-N’-neu-TSP-1 siRNA vaccination group. The expression of CD4+ subset Th1 cytokine-interferon-γ, increased in the groups of mice combing with TSP-1 siRNA. In order to eliminate the immune response induced by U6 promoter, we fused human-cyto-N’-neu with TSP-1 scramble siRNA as a control group to confirm the hypothesis accurately. We proved that the TSP-1 scramble siRNA reversed therapeutic efficacy and immune response compared with TSP-1 siRNA. In this thesis, we found that the therapeutic efficacy was improved by delivering TSP-1 siRNA into dendritic cells with gene gun. The mechanism of immune response induced by human-cyto-N’-neu-TSP-1 siRNA may due to down-regulate of Tregs and up-regulate of Th1.