PROLONGED SURVIVAL OF RAT LIVER ALLOGRAFTS TRANSFECTED WITH ADENOVIRUS VECTORS CONTAINNING FAS-LIGAND AND CRM A GENES

目的。fas配体（FasL）在角膜、睾丸等免疫特权部位逃避宿主免疫应答中起重要作用。细胞因子反应修饰因子A （CrmA）是牛痘病毒的基因产物，被认为可以阻断异体移植中Fas/FasL和穿孔素/颗粒酶介导的凋亡途径。我们在本研究中发现FasL和CrmA是延长大鼠同种异体肝移植存活的有效基因。方法。采用腺病毒载体和cre介导的基因传递系统在供肝中表达FasL和CrmA，免疫染色证实基因表达，体外诱导和抑制凋亡实验检测其功能。采用DA （RT-l<a>）与Lewis （RT-l^<l>）大鼠联合，采用FasL和/或CrmA基因转移的肝移植进行原位肝移植。结果。在cre介导的开关系统下成功制备了表达FasL （AxCALNLFasL）和CrmA （AxCALNLCrmA）的重组腺病毒载体，并通过免疫染色检测了其在转染细胞中的基因表达。COS-7细胞上表达FasL诱导Jurkat细胞凋亡；CrmA基因的表达可显著减少Hep2细胞的凋亡。将FasL和/或crma转染的DA肝脏移植到Lewis大鼠体内，可显著延长受体存活时间。结论。基于这些观察，我们得出结论，在供肝中FasL的适当表达水平可以诱导受体激活的T细胞凋亡，从而调节对同种异体肝移植的免疫反应。此外，CrmA是一种有效的基因产物，通过在基因转移的移植物中调节细胞凋亡活性来延长受体的生存时间。

Purpose. Fas-ligand (FasL) plays an important role in immune privileged sites including cornea and testis, to escape from host immune response. Cytokine response modifier A (CrmA), a gene product of cowpox virus, is considered to block Fas/FasL and perforin/granzyme mediated apoptotic pathways in allogeneic transplantation. We investigated in the present study, that FasL and CrmA are the effective genes to prolong survival of rat liver allografts. Methods. FasL and CrmA were expressed in donor liver using adenovirus vector with a Cre-mediated gene delivery system The gene expressions were confirmed by immune staining and their functions were examined with in vitro assay for induction and inhibition of the apoptosis. Using DA (RT-l<a>) to Lewis (RT-l^<l>) rat combination, orthotopic liver transplantation was performed with FasL and/or CrmA gene-transfered grafts. Results. The recombinant adenoviral vectors expressing FasL (AxCALNLFasL) and CrmA (AxCALNLCrmA) under a Cre-mediated switching system were successfully generated and their gene expressions were detected in the transfected cells by immune staining. FasL expressed on the COS-7 cells were induced apoptosis in the Jurkat cell ; CrmA gene-expression dramatically reduced apoptosis in Hep2 cells. The FasL and/or CrmA-transfected DA livers were transplanted into Lewis rats, resulting in a significant prolongation of recipient survival time. Conclusions. Based on these observations, we concluded that an appropriate expression level of FasL in donor liver could act to induce apoptosis to recipient activated T cells, which resulted in the regulation of immune response to the liver allograft. In addition, CrmA is an effective gene-product to prolong recipient survival by the apoptosis modulation activity in the gene-transferred graft.