| Objective:To establish an imaging system for evaluating the Caspase-3proteinase activity and monitoring the anti-caspase-3inhibitors by bioluminescence both in vitro and in vivo.Method:Based on the different parts of this project.First of all, in order to establish the in vitro system, which is the cellular-based model, using a reporter assay for imaging of Caspase-3protease activity in vitro.Taking split firefly luciferase complementation strategy, the reporter vector attB-ANluc (DEVD) BCluc was been constituted by the split N-and C-terminal fragments of luciferase, and fused to highly affinity peptides, pepA and pepB, respectively, and then inserted the Caspase-3cleavage sit of DEVD. While activating the Caspase-3proteinase, activated Caspase-3will cleave the reporter, enabling separation of ANLuc and BCLuc. Highly-affinity peptide A and peptide B lead to NLuc and CLuc complementation, and therefor restores luciferase activity.We cotransfected attB-ANluc (DEVD) BCluc into Hepal-6cell linage,48h later, cells were harvested and the luciferase activity was detected by bioluminescence imaging. The results proved that both exogenous and active Caspase-3protease cleaved the attB-ANluc (DEVD) BCluc reporter plasmid, which lead to an increased luciferase activity. In living animals, we cotransfected attB-ANluc (DEVD) BCluc through hydrodynamic tail vein injection and the luciferase activity was detected by bioluminescence imaging. We obtained identical results with that in cellular based model.Secondly, in order to establish the system in vivo, this is considered the live animal-based model.We tried to integrate the reporter expression cassette attB-ANLuc (DEVD) BCLuc into mouse liver chromosome and thus established a reporter mouse model that allows noninvasive detection of caspase-3activity in liver. The reporter plasmid attB-ANLuc (DEVD) BCLuc that contains fragment of attB and ANLuc (DEVD) BCLuc was codelivered with φpC31integrase plasmids specifically to mouse liver by hydrodynamic injection procedure.(?)C31integrase mediated integration of the reporter gene into mouse liver chromosome.Thirdly, we were thinking about how to apply for the cellular-based model and live mouse model to evaluate the different induced apoptosis conditions and scan different anti-apoptosis inhibitors. Specifically to say what we tested as follows:1. Under cellular-based model we tested one DEVD-targeted siRNA, one of anti-apoptosis inhibor (Z-VAD-FMK) and various concentrations of Dox to treat Hepal-6cells.2. Under mouse-based model we tested various liver damage conditions which can indicate the different clinicals.We used these mice to characterize in vivo activation of caspase-3upon treatment with ConA, LPS/GalN and infection with MHV; and then we tested the DEVD-targeted siRNA and one inhibor (Z-VAD-FMK).Results:The first part proved that both exogenous and active Caspase-3protease cleaved the attB-ANluc (DEVD) BCluc reporter plasmid, which leads to an increase in luciferase activity, in consistent with the results from Western blot; we saw the active Caspase-3protein cleaved the fused flue protein.Thesecond part is through nested PCR assay, bioluminescent technique and western blot assay showed that the established mouse model can be used for monitering various liver damaged conditions and as well for screening anti-apoptosis compounds.The third part reached the results as follows:1. The result showed, the reporter assay system using split firefly luciferase complementation strategy proved useful for evaluating Caspase-3protease activity in cells level.2. Our data showed that liver apoptosis can be directly monitored by our mouse model through imaging the activity of luciferase; and shRNA targeting caspase-3protein and anti-apoptosis inhibitors were also been effectively evaluated. ConclusionOur data showed that liver apoptosis could be evaluated by the activity of luciferase in vitro and in vivo model. The models we set up can be used for screening anti-apoptosis compounds targeted to liver cell apoptosis. |
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