| Summary: | 碩士 === 國立成功大學 === 生物化學研究所 === 92 === Thrombomodulin (TM) is a glycoprotein that was originally identified on vascular endothelium and is well-characterized as a natural endothelial anticoagulant factor. Studies by several investigators have elucidated multiple aspects of TM functions in fibrinolysis, inflammation, proliferation, and in embryogenesis. Functional evaluation of TM in the epithelium cell line HaCaT cells was performed by using RNA interference (RNAi) in this study. RNAi is a sequence-specific post-transcriptional gene silencing mechanism mediated by double-stranded RNA (dsRNA) molecules. It has become a popular research tool in annotating gene function. We used the vector coding a hairpin siRNA against TM gene under the control of mammalian polymerase III U6 promoter. The initial analysis of TM siRNA efficiency was performed in TM negative A2058 cells co-transfected with siRNA and a TM-expressing construct. The best candidate of siRNA that could effectively suppress TM expression was selected and used in the study on the function of HaCaT cells. The endogenous TM level was markedly reduced by siRNA. Protein C activation assay revealed that siRNA diminished TM expression and reduced the TM activity on the cell surface. Moreover, knockdown of TM expression increased the HaCaT cell proliferation and migration, but not in mock cells with control plasmid. These results indicated that siRNA-mediated gene silencing of endogenous TM transcripts has the potential applications for studying TM functions in the epithelium keratinocytes.
In another way, we used a commercially adenoassociated viral (AAV) vector for siRNA delivery into mammalian cells. The recombinant AAV vector expresses the enhanced green fluorescent protein (EGFP) under the control of CMV promoter and a U6 promoter-driven hairpin siRNA against TM gene. Recombinant AAV infectious viral particles were produced successfully in 293 cells by cotransfecting three plasmids containing pAAV-EGFP vector genes, adenovirus helper genes, and a vector genome pAAV-RC (replication and capsid). Significantly high level of infection efficiency was achieved by examining the EGFP expression in HeLa cells after infection. We will further apply the infectious virus to deliver siRNA into other TM-expressed cells for studying the function of TM.
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