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

在研究的第一年，我们使用mCAP RNA Capping Kit成功地稳定合成和扩增了mRNA。接下来，我们使用HVJ Envelope VECTOR KIT进行mRNA的体外转移研究，BHK21细胞的荧光素酶检测结果比对照组提高了500 ~ 1000倍。将含荧光素酶mRNA的HVJ包膜载体经门静脉注射到C57BL/6小鼠体内肝脏，可获得5 ~ 10倍的增加。第二年，我们采用同样的方法，在离体4C时通过门静脉向Wistar大鼠肝脏注射mRNA。然而，在荧光素酶测定中仅观察到2至3倍的增加。因此，我们尝试用回声对比微泡（option）代替HVJ包膜载体进行超声介导的基因转移，该方法最近被证明是高效的。在使用荧光素酶报告基因的初步实验中，转染和同基因移植后24小时的肝移植物荧光素酶活性比单独输注质粒显著提高约17倍。当血管夹紧同时进行时，获得额外的10倍增加。下一步将采用超声介导的方法将mRNA转移到肝移植物中。

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.