Suppression of hepatic ischemia/reperfusion injury by early expression of protective protein

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In the first year of the study period, we succeeded in stable synthesis and amplification of mRNA using mCAP RNA Capping Kit. Next, we moved to in vitro mRNA transfer study using HVJ Envelope VECTOR KIT and got 500- to 1000-fold increase in luciferase assay in BHK21 cells compared with control. When HVJ envelope vector containing luciferase mRNA was injected to the liver via portal vein in C57BL/6 mice in vivo, 5- to 10-fold increase was obtained. In the second year, we injected mRNA to the liver via portal vein in Wistar rats at 4C ex vivo using the same method. However, only 2- to 3-fold increase was observed in luciferase assay. We, therefore, tried ultrasound-mediated gene transfer with echo contrast microbubble (Optison), which has recently been shown to be highly efficient, in stead of HVJ envelope vector. In the preliminary experiment using luciferase reporter gene, luciferase activity of liver graft 24 hours after transfection and syngeneic transplantation was significantly increased about 17-fold as compared with plasmid infusion alone. When vascular clamping was performed together, additional 10-fold increase was obtained. In the next step, mRNA transfer into liver graft using ultrasound-mediated method would be performed.