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

在第一年的研究期间，我们成功地稳定合成和扩增的mRNA使用mCAP RNA加帽试剂盒。接下来，我们使用HVJ Envelope VECTOR KIT进行体外mRNA转移研究，与对照相比，BHK 21细胞中的荧光素酶测定增加了500至1000倍。当将含有荧光素酶mRNA的HVJ包膜载体通过门静脉注射到C57 BL/6小鼠体内的肝脏中时，获得了5至10倍的增加。在第二年，我们使用相同的方法在4C离体条件下将mRNA通过门静脉注射到Wistar大鼠的肝脏中。然而，在荧光素酶测定中仅观察到2至3倍的增加。因此，我们尝试用超声造影微泡（Optison）代替HVJ包膜载体进行超声介导的基因转移，该方法最近已被证明是高效的。在使用荧光素酶报告基因的初步实验中，与单独质粒输注相比，转染和同基因移植后24小时的肝移植物的荧光素酶活性显著增加约17倍。当同时进行血管夹闭时，获得额外的10倍增加。下一步，将采用超声介导的方法将mRNA转移到移植肝中。

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.